USOO9714897B2

(12) United States Patent (10) Patent No.: US 9,714,897 B2 Kim et al. (45) Date of Patent: Jul. 25, 2017

(54) HYDROGEL PARTICLES WITH TUNABLE (58) Field of Classification Search OPTICAL PROPERTIES AND METHODS None FOR USING THE SAME See application file for complete search history. (71) Applicant: SLINGSHOT BIOSCIENCES, INC., Emeryville, CA (US) (56) References Cited U.S. PATENT DOCUMENTS (72) Inventors: Jeffrey Kim, Berkeley, CA (US); Oliver Liu, San Francisco, CA (US); 4,704,891 A 11/1987 Recktenwald et al. Jeremy Agresti, El Cerrito, CA (US); 4,774,189 A 9, 1988 Schwartz Anh Tuan Nguyen, San Jose, CA (US) (Continued) (73) Assignee: Slingshot Biosciences, Inc., Emeryville, FOREIGN PATENT DOCUMENTS CA (US) WO 89.10566 11, 1989 (*) Notice: Subject to any disclaimer, the term of this WO OOO8212 2, 2000 patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 15/018,769 Ugelstad, J. and Mork, P.C., “Swelling of Oligomer-Polymer Par (22) Filed: Feb. 8, 2016 ticles. New Methods of Preparation of Emulsions and Polymer Dispersions.” Advances in Colloid and Interface Sciences, 13 (65) Prior Publication Data (1980), pp. 101-140. US 2016/O25885.6 A1 Sep. 8, 2016 (Continued) Related U.S. Application Data Primary Examiner — Robert Xu (60) Provisional application No. 62/114,004, filed on Feb. (74) Attorney, Agent, or Firm — Cooley LLP. Nathan W. 9, 2015, provisional application No. 62/184,192, filed Poulsen on Jun. 24, 2015. (57) ABSTRACT (51) Int. C. Hydrogel particles and their use in cytometic applications GOIN 3L/00 (2006.01) are described. The hydrogel particles described herein are GOIN 15/10 (2006.01) selectively tunable to have at least one optical property (Continued) Substantially similar to the at least one optical property of a (52) U.S. C. target cell. In this regard, the hydrogel particles provided CPC ...... G0IN 15/1012 (2013.01): CI2O I/04 herein in one aspect, are used as a calibration reagent for the (2013.01); G0IN I/28 (2013.01); G0IN detection of a target cell in a sample. 15/1434 (2013.01); (Continued) 26 Claims, 13 Drawing Sheets

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(51) Int. Cl. FOREIGN PATENT DOCUMENTS CI2O I/04 (2006.01) WO O 132829 5, 2001 GOIN I/28 (2006.01) WO 03000014 A2 1/2003 GOIN 15/14 (2006.01) WO 2008115653 A2 9, 2008 GOIN 1.5/OO (2006.01) WO 2010O25988 A1 3f2010 (52) U.S. Cl. WO 20110984O7 A1 8, 2011 CPC. G0IN 15/1459 (2013.01); G0IN 2001/2893 WO 2012033811 A1 3f2012 (2013.01); G0IN 2015/0065 (2013.01); G0IN 2015/1006 (2013.01); G0IN 2015/1018 OTHER PUBLICATIONS (2013.01); G0IN 2015/149 (2013.01) Lee, Ki-Chang and Lee, Sang-Yun, “Preparation of Highly Cross Linked, Monodisperse Poly(methyl methacrylate) Microspheres by (56) References Cited Dispersion Polymerization; Part II Semi-continuation Processes.” U.S. PATENT DOCUMENTS Macromolecular Research, vol. 16, No. 4, (2008) pp. 293-302. Jin-Woong Kim, Andrew S. Utada, Alberto Fernandez-Nieves, 5,093,234 A 3, 1992 Schwartz Zhibing Hu, and David A.Weitz, "Fabrication of Monodisperse Gel 5,283,079 A 2, 1994 Wang et al. Shells and Functional Microgels in Microfluidic Devices.” Angew. 5,395,688 A 3, 1995 Wang et al. Chem. Int. Ed. (2007) 46, pp. 1819-1822. 5,820,879 A 10, 1998 Fernandez et al. International Search Report and Written Opinion for International 6,586, 176 B1 T/2003 Trnovsky et al. Patent Application No. PCT/US2016/017029, mailed May 19, 6,806,058 B2 10, 2004 Jesperson et al. 2016. RE39,542 E 4, 2007 Jain et al. Patanarut, Alexis, et al. “Synthesis and characterization of hydrogel 7,314,584 B2 1, 2008 Tsutsui et al. particles containing Cibacron Blue F3G-A.” Colloids and Surfaces 8,030,095 B2 10, 2011 Harriman A: Physicochemical and Engineering Aspects 362. 1 (2010): 8-19. 8, 187,885 B2 5, 2012 Purvis, Jr. Luchini, Alessandra, et al. “Smart hydrogel particles: biomarker 8,415,173 B2 4, 2013 Harriman harvesting: one-step affinity purification, size exclusion, and pro 8,704,158 B2 4, 2014 Haberstroh et al. tection against degradation.” Nano letters 8.1 (2008): 350-361. 2003/O132538 A1 T/2003 Chandler Bele, Marjan, Olavi Siiman, and Egon Matijevic. “Preparation and 2005/0176056 A1 8, 2005 Sammak et al. flow cytometry of uniform silica-fluorescent dye microspheres.” 2006/024.0560 A1 10, 2006 Bakker et al. Journal of colloid and interface science 254.2 (2002): 274-282. 2007/0054119 A1 3, 2007 Garstecki et al. Proff, Guenther, et al. "Potential of label-free detection in high 2007/025.9415 A1 11/2007 Zigova et al. content-screening applications.” Journal of Chromatography A 2009, O14896.1 A1 6, 2009 Luchini et al. 2010/023.4252 A1 9, 2010 Moradi-Araghi et al. 1161.1 (2007): 2-8. 2010/028.5594 A1* 11, 2010 Purvis, Jr...... GON 15, 1012 Hasegawa, Urara, et al. “Nanogel-quantum dot hybrid nanoparticles 436/10 for live cell imaging.” Biochemical and biophysical research com 2011/0318820 12/2011 Hinz et al. munication 331.4 (2005): 917-921. 2012/0309651 12, 2012 Pregibon ...... C12O 1/6816 Tomczak, Nikodem, et al. “Designer polymer-quantum dot archi 506/16 tectures. Progress in Polymer Science 34.5 (2009): 393-430. 2013/O177973 A1 T/2013 Kondo 2015 O1771.15 A1 6, 2015 Kim et al. * cited by examiner U.S. Patent Jul. 25, 2017 Sheet 1 of 13 US 9,714.897 B2

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Fig. 13 US 9,714,897 B2 1. 2 HYDROGEL PARTICLES WITH TUNABLE method further comprises measuring the at least one optical OPTICAL PROPERTIES AND METHODS property of the hydrogel particle using the cytometric FOR USING THE SAME device. A sample comprising a plurality of cells is inserted into the cytometric device, and the at least one optical CROSS REFERENCE TO RELATED property of individual cells of the plurality are measured. APPLICATIONS Finally, a determination is made, based on the optical property measurement, whether the target cell or plurality This application claims priority from U.S. Provisional thereof is present in the sample. Application No. 62/114,004, filed Feb. 9, 2015 and U.S. In one embodiment of the methods provided herein, the Provisional Application No. 62/184,192, filed Jun. 24, 2015, 10 hydrogel particle comprises a biodegradable monomer. In a the disclosure of each of which is incorporated by reference further embodiment, the biodegradable monomer is a mono herein in their entireties. saccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. In even a further embodiment, the BACKGROUND OF THE INVENTION biodegradable monomer is functionalized with acrylamide 15 or acrylate. Flow cytometry is a technique that allows for the rapid separation, counting, and characterization of individual cells BRIEF DESCRIPTION OF THE FIGURES and is routinely used in clinical and laboratory settings for a variety of applications. The technology relies on directing FIG. 1 illustrates the optical properties of disclosed a beam of light onto a hydrodynamically-focused stream of hydrogel particles compared to polystyrene beads. liquid. A number of detectors are then aimed at the point FIG. 2 depicts the process of producing labeled hydrogel where the stream passes through the light beam: one in line particles of the disclosure. with the light beam (Forward Scatter or FSC) and several FIG. 3 provides brightfield and fluorescent images of perpendicular to it (Side Scatter or SSC). FSC correlates labeled hydrogel particles of the disclosure. with the cell volume and SSC depends on the inner com 25 FIG. 4 illustrates the use of hydrogel particles of the plexity of the particle (e.g., shape of the nucleus, the amount disclosure as calibrants for cell types displaying a variety of and type of cytoplasmic granules or the membrane rough optical scattering properties. ness). As a result of these correlations, different specific cell FIG. 5 provides dating showing correlation of inter-drop types exhibit different FSC and SSC, allowing cell types to delay for a flow cytometer with hydrogel particle diameter. be distinguished in flow cytometry. 30 FIG. 6 provides brightfield (6A and 6C) and fluorescent The ability to identify specific cell types, however, relies (6B and 6D) images of Chinese Hamster Ovary cells (6A on proper calibration of the instrument, a process that has and 6B) and hydrogel particles of the disclosure (6C and relied on the use of purified cells of the cell type of interest. 6D). Obtaining these purified cells can require costly, laborious FIG. 7 provides data showing comparison of human procedures that are prone to batch-to-batch variation. There 35 buccal cells to hydrogel particles encapsulating different fore, there is a need in the art for synthetic compositions with amounts of DNA, as measured by fluorescence-activated tunable optical properties that can mimic specific cell types cell sorting (FACS). in devices such as flow cytometers. FIG. 8 provides data for hydrogel particles encapsulating nanoparticles at different concentrations, demonstrating tun SUMMARY OF THE INVENTION 40 ing of side scattering independent of forward Scattering. FIG. 9 provides data for hydrogel particles produced with In one aspect of the invention, a hydrogel particle com different percentages of polymer, demonstrating tuning of prising a polymerized monomer and having at least one refractive index measured by forward Scattering. Surface is provided. The hydrogel particle has at least one FIG. 10 shows one embodiment of hydrogel parameter optical property that is substantially similar to the at least 45 tuning to match and/or mimic desired cell population met one optical property of a target cell. The optical property in 1CS one embodiment, is a side scatter profile (SSC), forward FIGS. 11 and 12 are diagrams showing embodiments of scatter profile (FSC), a fluorescence emission profile, or a how to adjust the forward Scatter, side scatter and Surface combination thereof. The target cell can be any target cell properties of a hydrogel particle. that the user specifies. For example, in one embodiment, the 50 FIG. 13 are scatter plots for various hydrogel particles (A) target cell is an immune cell, stem cell or cancer cell. and (B) and a commercial blood sample (C). In another aspect, a method for calibrating a cytometric device for analysis of a target cell, is provided. In one DETAILED DESCRIPTION OF THE embodiment, the method comprises inserting into the device INVENTION a hydrogel particle having at least one optical property 55 Substantially similar to a target cell, wherein the hydrogel The indefinite articles “a” and “an and the definite article particle comprises a polymerized monomer and has at least “the are intended to include both the singular and the plural, one surface. The method further comprises measuring the at unless the context in which they are used clearly indicates least one optical property of the hydrogel particle using the otherwise. cytometric device. The at least one optical property in one 60 “At least one' and “one or more are used interchange embodiment, is used as a reference to detect a target cell in ably to mean that the article may include one or more than a sample. one of the listed elements. In yet another aspect, a method for detecting a target cell Unless otherwise indicated, it is to be understood that all in a sample is provided. The method comprises inserting into numbers expressing quantities, ratios, and numerical prop the device a hydrogel particle having at least one optical 65 erties of ingredients, reaction conditions, and so forth, used property Substantially similar to a target cell, wherein the in the specification and claims are contemplated to be able hydrogel particle comprises a polymerized monomer. The to be modified in all instances by the term “about'. US 9,714,897 B2 3 4 Several critical calibration measurements for flow cytom cussed further below, the use of bifunctional monomers eters require precise time resolution, Such as setting the allows for the further derivatization of hydrogels, e.g., with offset time between lasers, and calculating the delay time fluorescent dyes, cell Surface markers or epitope binding between detection and sorting of an object. Due to the fluidic fragments thereof, or a combination thereof. An example of conditions within the instrument, precise setting of these hydrogel parameter tuning to meet/match desired cell Sub timing parameters requires the use of calibration particles population metrics is provided at FIG. 10. Methods for that are the same size as the cells to be analyzed. Timing tuning the properties of a hydrogel are described herein. The calibrations are typically performed using polystyrene beads ability to adjust a range of parameters including hydrogel with variable fluorescent intensities to calibrate the response components and concentration of the same allows for the of an excitation source and to set the inter-laser timing delay 10 ability to tune a particle to mimic a wide range of cells, for and Sorting delay. Flow cytometers can also be calibrated example one of the cell types described herein. using forward and side scatter signals which are general As provided above, in one aspect, the present invention measures of size and granularity or complexity of the target provides individual hydrogel particles each having one or sample. These calibrations are crucial for the accurate per more optical properties substantially similar to one or more formance of the cytometer and for any downstream analysis 15 optical properties of a target cell. In one embodiment, the or sorting of cell populations. The disclosed hydrogel par one or more optical properties, is a side scatter profile, a ticles exhibit tuned scatter properties and are suitable for use forward scatter profile or a secondary marker profile. Such as as calibration reagents for a range of mammalian or bacterial a fluorescence marker profile, for example a fluorescence cell types. Scattering is a standard metric for distinguishing marker profile of a fluorescently-labeled antibody that binds cell types in heterogeneous mixtures for clinical, food safety, to the surface of the hydrogel particle. “Substantially simi and research purposes. lar, as used herein, denotes at least 40% similar, at least Although polystyrene particles can be used to set inter 50% similar, at least 60% similar, at least 70% similar, at laser and sorting delays for Some applications, many eukary least 80% similar, at least 90% similar, at least 95% similar, otic cell types fall outside of the size range of commercially at least 96% similar, at least 97% similar, at least 98% available polystyrene particles (1-20 um) making it nearly 25 similar or at least 99% similar. impossible to accurately calibrate a flow cytometer for these The present invention is based in part on the unexpected targets. Also, as shown in FIG. 1, polystyrene particles are discovery that one or more optical properties of a hydrogel fundamentally limited in the optical properties that can particle can be independently modulated by altering the possess Such as side scattering, which is a general measure composition of the hydrogel particle, for example, by alter of cellular complexity. Polystyrene particles are therefore 30 ing the amount of initial monomer (or co-monomer) in the limited in the two most important passive optical measure composition, by altering the Surface functionalization, by ments used in flow cytometry: FSC (forward scattering), and altering the amount of a polymerization initiator or by SSC (side scattering) which measure the size and complex altering the amount of crosslinker. For example, side scat ity of the target respectively. Due to these limitations of tering (SSC) can be modulated without substantially affect polystyrene, users must rely on purified cell lines to calibrate 35 ing forward scattering (FSC), and vice versa. Furthermore, fluorescent intensity, inter-laser delay, sort delays, size and the optical properties (e.g. refractive index) of hydrogel cellular complexity for experiments. This is a lengthy and particles can be tuned without having a Substantial effect on labor-intensive process that increases the cost of flow density of the particle. This is a Surprising and useful feature, cytometry validation and research pipelines significantly. as hydrogel particles that serve as Surrogates for cells in More importantly, these calibration cell lines introduce 40 cytometric methods such as flow cytometry or (fluores biological variation, causing disparities in the interpretation cence-activated cell sorting) FACS require a minimal den of data. sity in order to function in those assays. Moreover, quality control (QC) for calibration of flow In another aspect, a method for producing a hydrogel cytometers is also a crucial consideration when these instru particle is provided, wherein the hydrogel particle has one or ments are used for clinical applications, for example, to 45 more optical properties Substantially similar to the optical isolate human T-regulatory cells or stem cells for down properties of one or more target cells. In one embodiment, stream cellular therapies. The FDA mandates that the ste the hydrogel particle has pre-determined optical properties. rility, identity, purity, and potency of a cell therapy product The optical property, in one embodiment, is SSC, FSC, be demonstrated before administration to patients (Riley et fluorescence emission, or a combination thereof. al. (2009). Immunity 30, pp. 656-665). Contamination of a 50 In yet another aspect, a method of calibrating a cytometric cellular population with polystyrene QC particles could device for analysis of a target cell is provided. In one therefore be problematic, as polystyrene has been implicated embodiment, the method comprises (a) inserting into the in certain cancers. Additionally, a cellular population that is device a hydrogel particle having optical properties Substan contaminated with a QC standard that is enzymatically tially similar to the optical properties of the target cell; b) degraded or digested internally after administration to a 55 measuring the optical properties of the hydrogel particle patient potentially overcomes contamination issues, should using the cytometric device, thereby calibrating the cyto they arise. metric device for analysis of the target cell. Cytometric The present invention addresses these and other needs, as devices are known in the art, and include commercially discussed below. available devices for performing flow cytometry and FACS. In one aspect, a composition comprising a plurality of 60 As provided above, in one aspect of the invention, com hydrogel particles is provided, wherein the individual hydro positions comprising a plurality of hydrogel particles are gel particles of the plurality each has one or more optical provided. A hydrogel is a material comprising a macromo properties Substantially similar to one or more optical prop lecular three-dimensional network that allows it to swell erties of a target cell. Each of the individual hydrogel when in the presence of water, to shrink in the absence of (or particles of the plurality independently comprises a hydrogel 65 by reduction of the amount of) water, but not dissolve in which is synthesized by polymerizing one or more mono water. The Swelling, i.e., the absorption of water, is a mers, i.e., to form a homopolymer or copolymer. As dis consequence of the presence of hydrophilic functional US 9,714,897 B2 5 6 groups attached to or dispersed within the macromolecular ence in its entirety for all purposes. For example, hydrazine network. Crosslinks between adjacent macromolecules (e.g., with an NHS ester compound) or EDC coupling result in the aqueous insolubility of these hydrogels. The reactions (e.g., with a maleimide compound) can be used to cross-links may be due to chemical (i.e., covalent) or physi construct the hydrogels of the invention. cal (i.e., VanDer Waal forces, hydrogen-bonding, ionic In one embodiment, a monomer for use with the hydro forces, etc.) bonds. Synthetically prepared hydrogels can be gels provided herein is lactic acid, glycolic acid, acrylic acid, prepared by polymerizing a monomeric material to form a 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hy backbone and cross-linking the backbone with a crosslink droxyethyl methacrylate (HEMA), propylene glycol meth ing agent. As referred to herein, the term “hydrogel” refers acrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl to the macromolecular material whether dehydrated or in a 10 methacrylate, glycidyl methacrylate, glycerol methacrylate hydrated state. A characteristic of a hydrogel that is of (GMA), glycol methacrylate, ethylene glycol, fumaric acid, particular value is that the material retains the general shape, a derivatized version thereof, or a combination thereof. whether dehydrated or hydrated. Thus, if the hydrogel has an In one embodiment, one or more of the following mono approximately spherical shape in the dehydrated condition, mers is used herein to form a hydrogel of the present it will be spherical in the hydrated condition. 15 invention: 2-hydroxyethyl methacrylate, hydroxyethoxy In one embodiment, a hydrogel particle disclosed herein ethyl methacrylate, hydroxydiethoxyethyl methacrylate, comprises greater than about 30%, greater than about 40%, methoxyethyl methacrylate, methoxyethoxyethyl methacry greater than about 50%, greater than about 55%, greater than late, methoxydiethoxyethyl methacrylate, poly(ethylene about 60%, greater than about 65%, greater than about 70%, glycol) methacrylate, methoxy-poly(ethylene glycol) meth greater than about 75%, greater than about 80%, greater than acrylate, methacrylic acid, sodium methacrylate, glycerol about 85%, greater than about 90%, or greater than about methacrylate, hydroxypropyl methacrylate, hydroxybutyl 95% water. In another embodiment, a hydrogel particle has methacrylate or a combination thereof. a water content of about 10 percent by weight to about 95 In another embodiment, one or more of the following percent by weight, or about 20 percent by weight to about 95 monomers is used herein to form a tunable hydrogel: phenyl percent by weight, or about 30 percent by weight to about 95 25 acrylate, phenyl methacrylate, benzyl acrylate, benzyl meth percent by weight, or about 40 percent by weight to about 95 acrylate, 2-phenylethyl acrylate, 2-phenylethyl methacry percent by weight, or about 50 percent by weight to about 95 late, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, percent by weight, or about 60 percent by weight to about 95 phenylthioethyl acrylate, phenylthioethyl methacrylate, 2.4. percent by weight, or about 70 percent by weight to about 95 6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacry percent by weight, or about 80 percent by weight to about 95 30 late, pentabromophenyl acrylate, pentabromophenyl meth percent by weight. acrylate, pentachlorophenyl acrylate, pentachlorophenyl The hydrogels provided herein, in the form of particles, methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopro are synthesized by polymerizing one or more of the mono pyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacry mers provided herein. The synthesis is carried out to form late, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacry individual hydrogel particles. The monomeric material 35 late, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl (monomer) in one embodiment is polymerized to form a methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophe homopolymer. However, in another embodiment copoly noxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, mers of different monomeric units (i.e., co-monomers) are 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, synthesized and used in the methods provided herein. The N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl monomer or co-monomers used in the methods and com 40 methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl positions described herein, in one embodiment, is a bifunc methacrylamide, N-diphenylmethyl acrylamide N-(4-meth tional monomer or includes a bifunctional monomer (where ylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, co-monomers are employed). In one embodiment, the N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, hydrogel is synthesized in the presence of a crosslinker. In N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acryl a further embodiment, embodiment, the hydrogel is synthe 45 amide, N.N-methylphenyl acrylamide, N.N-phenyl phenyl sized in the presence of a polymerization initiator. ethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4- The amount of monomer can be varied by the user of the methyl phenyl)methyl methacrylamide, N-1-naphthyl invention, for example to obtain a particular optical property methacrylamide, N-4-nitrophenyl methacrylamide, N-(2- that is Substantially similar to that of a target cell. In one phenylethyl)methacrylamide, N-triphenylmethyl methacry embodiment, the monomeric component(s) (i.e., monomer, 50 lamide, N-(4-hydroxyphenyl)methacrylamide, N.N-methyl co-monomer, bifunctional monomer, or a combination phenyl methacrylamide, N,N'-phenyl phenylethyl thereof, for example, bis/acrylamide in various crosslinking methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vi ratios, allyl amine or other co-monomers which provide nylpyridine, as described in U.S. Pat. No. 6,657,030, which chemical functionality for secondary labeling/conjugation or is incorporated by reference in its entirety herein for all alginate is present at about 10 percent by weight to about 95 55 purposes. percent weight of the hydrogel. In a further embodiment, the Both synthetic monomers and bio-monomers can be used monomeric component(s) is present at about 15 percent by in the hydrogels provided herein, to form synthetic hydro weight to about 90 percent weight of the hydrogel, or about gels, bio-hydrogels, or hybrid hydrogels that comprise a 20 percent by weight to about 90 percent weight of the synthetic component and a bio-component (e.g., peptide, hydrogel. 60 protein, monosaccharide, disaccharide, polysaccharide, pri Examples of various monomers and cross-linking chem mary amines Sulfhydryls, carbonyls, carbohydrates, carbox istries available for use with the present invention are ylic acids present on a biomolecule). For example, proteins, provided in the Thermo Scientific Crosslinking Technical peptides or carbohydrates can be used as individual mono Handbook entitled “Easy molecular bonding crosslinking mers to form a hydrogel that includes or does not include a technology.” (available at tools.lifetechnologies.com/con 65 synthetic monomer (or polymer) and in combination with tent/sfs/brochures/1602163-Crosslinking-Reagents-Hand chemically compatible co-monomers and crosslinking book.pdf, the disclosure of which is incorporated by refer chemistries (see for example, the Thermo Scientific Cross US 9,714,897 B2 7 8 linking Technical Handbook entitled “Easy molecular bond mers, and mixed compositions is obvious to those skilled in ing crosslinking technology, available at tools.lifetechnolo the art and follows chemical reactivity principles know to gies.com/content/sfs/brochures/1602163-Crosslinking those skilled in the art. (reference Thermo handbook and Reagents-Handbook.pdf, the disclosure of which is acrylamide polymerization handbook). See, for example, the incorporated by reference in its entirety for all purposes.). Thermo Scientific Crosslinking Technical Handbook Compatible crosslinking chemistries include, but are not entitled "Easy molecular bonding crosslinking technology,” limited to, amines, carboxyls, and other reactive chemical (available at tools.lifetechnologies.com/content/sfs/bro side groups. Representative reactive groups amenable for chures/1602163-Crosslinking-Reagents-Handbook.pdf) and use in the hydrogels and monomers described herein are the Polyacrylamide Emulsions Handbook (SNF Floerger, 10 available at Snf.com.au/downloads/Emulsion Hand provided in Table 1, below. book E.pdf), the disclosure of each of which is incorporated by reference in its entirety for all purposes. TABLE 1. In one embodiment, a hydrogel particle provided herein Crosslinker reactive groups amenable for bio-monomer conjugation comprises a polymerizable monofunctional monomer and is 15 a monofunctional acrylic monomer. Non-limiting examples Target of monofunctional acrylic monomers for use herein are functional acrylamide; methacrylamide, N-alkylacrylamides such as Reactivity class group Reactive chemical group N-ethylacrylamide, N-isopropylacrylamide or N-tertbuty Amine reactive —NH2 NHS ester lacrylamide: N-alkylmethacrylamides such as N-ethylmeth midoester Penafluorophenyl ester acrylamide or Nisopropylmethacrylamide: N,N-dialkylacry Hydroxymethyl phosphine lamides such as N,N-dimethylacrylamide and N,N-diethyl Carboxyl-to-amine reactive —COOH Carbodiimide (e.g., EDC) acrylamide, N-dialkylamino)alkylacrylamides such as Sulfhydryl-reactive - SH Maeleimide N-3dimethylamino) propylacrylamide or N-3-(diethyl Haloacetyl (bromo- or amino)propylacrylamide, N-(dialkylamino) alkylmeth iodo-) Pyridyllisulfide 25 acrylamides such as N-3-dimethylamino)propyl)methacry Thiosulfonate lamide or N-3-(diethylamino) propyl)methacrylamide: Vinylsulfonate (dialkylamino)alkyl acrylates such as 2-(dimethylamino) Aldehyde-reactive (oxidized - CHO Hydrazine Sugars, carbonyls) Alkoxyamine ethyl acrylate, 2-(dimethylamino)propyl acrylate, or 2-(di Photo-reactive, i.e., Random Diazirine ethylamino)ethyl acrylates; and (dialkylamino) alkyl meth nonselective, random insertion Aryl azide 30 acrylates such as 2-(dimethylamino)ethyl methacrylate. Hydroxyl (nonaqueous)-reactive —OH Socyanate A bifunctional monomer is any monomer that can polym Azide-reactive - N3 phosphine erize with a monofunctional monomer of the disclosure to form a hydrogel as described herein that further contains a In general, any form of polymerization chemistry/meth second functional group that can participate in a second ods commonly known by those skilled in the art, can be 35 reaction, e.g., conjugation of a fluorophore or cell Surface employed to form polymers. In some embodiments, polym receptor (or domain thereof). erization can be catalyzed by ultraviolet light-induced radi In some embodiments, a bifunctional monomer is selected cal formation and reaction progression. In other embodi from the group consisting of allyl amine, allyl alcohol, allyl ments, a hydrogel particle of the disclosure is produced by isothiocyanate, allyl chloride, and allyl maleimide. the polymerization of acrylamide or the polymerization of 40 A bifunctional monomer can be a bifunctional acrylic acrylate. For example, the acrylamide in one embodiment is monomer. Non-limiting examples of bifunctional acrylic a polymerizable carbohydrate derivatized acrylamide as monomers are N,N'-methylenebisacrylamide, N.N'methyl described in U.S. Pat. No. 6,107,365, the disclosure of which ene bismethacrylamide, N,N'-ethylene bisacrylamide, N,N'- is incorporated by reference in its entirety for all purposes. ethylene bismethacrylamide, N.N"propylenebisacrylamide As described therein and known to those of ordinary skill in 45 and N,N'-(1,2-dihydroxyethylene) bisacrylamide. the art, specific attachment of acrylamide groups to Sugars is Higher-order branched chain and linear co-monomers can readily adapted to a range of monosaccharides and higher be substituted in the polymer mix to adjust the refractive order polysaccharides, e.g., synthetic polysaccharides or index while maintaining polymer density, as described in polysaccharides derived from natural Sources, such as gly U.S. Pat. No. 6,657,030, incorporated herein by reference in coproteins found in serum or tissues. 50 its entirety for all purposes. In one embodiment, an acrylate-functionalized poly(eth In some embodiments, a hydrogel comprises a molecule ylene) glycol monomer is used as a hydrogel monomer. For that modulates the optical properties of the hydrogel. Mol example, the PEG in one embodiment is an acrylate or ecules capable of altering optical properties of a hydrogel acrylamide functionalized PEG. are discussed further below. In some embodiments, a hydrogel particle comprises a 55 In one embodiment, an individual hydrogel particle or a monofunctional monomer polymerized with at least one plurality thereof comprises a biodegradable polymer as a bifunctional monomer. One example includes, but is not hydrogel monomer. In one embodiment, the biodegradable limited to, the formation of poly-acrylamide polymers using polymer is a poly(esters) based on polylactide (PLA), acrylamide and bis-acrylamide (a bifunctional monomer). In polyglycolide (PGA), polycaprolactone (PCL), and their another embodiment, a hydrogel particle provided herein 60 copolymers. In one embodiment, the biodegradable polymer comprises a bifunctional monomer polymerized with a sec is a carbohydrate or a protein, or a combination thereof. For ond bifunctional monomer. One example include, but is not example, in one embodiment, a monosaccharide, disaccha limited to, the formation of polymers with mixed composi ride or polysaccharide, (e.g., glucose. Sucrose, or maltodex tion containing compatible chemistries Such as acrylamide, trin) peptide, protein (or domain thereof) is used as a bis-acrylamide, and bis-acrylamide structural congeners 65 hydrogel monomer. Other biodegradable polymers include containing a wide range of additional chemistries. The range poly(hydroxyalkanoate)s of the PHB-PHV class, additional of chemically compatible monomers, bifunctional mono poly(ester)S, and natural polymers, for example, modified US 9,714,897 B2 10 poly(saccharide)S. e.g., starch, cellulose, and chitosan. In 91-631-4985-0, the disclosure of each of which are hereby another embodiment, the biocompatible polymer is an adhe incorporated by reference in their entirety. sion protein, cellulose, a carbohydrate, a starch (e.g., malto Biocompatible monomers for use with the hydrogels dextrin, 2-hydroxyethyl starch, alginic acid), a dextran, a described herein include in one embodiment, ethyleglycol lignin, a polyaminoacid, an amino acid, or chitin. Such dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate biodegradable polymers are available commercially, for (HEMA), methylmethacrylte (MMA), methacryloxymeth example, from Sigma Aldrich (St. Louis, Mo.). yltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidon The protein in one embodiment comprises only natural (N-VP), styrene, or a combination thereof. amino acids. However, the invention is not limited thereto. Naturally occurring hydrogels useful in this invention For example, self-assembling artificial proteins and proteins 10 include various polysaccharides available from natural with non-natural amino acids (e.g., those incorporated into Sources such as plants, algae, fungi, yeasts, marine inverte non-ribosomal peptides or synthetically introduced via Syn brates and arthropods. Non-limiting examples include aga thetic approaches, see for example, Zhang et al. (2013). rose, dextrans, chitin, cellulose-based compounds, starch, Current Opinion in Structural Biology 23, pp. 581-587, the 15 derivatized starch, and the like. These generally will have disclosure of which is incorporated by reference in its repeating glucose units as a major portion of the polysac entirety for all purposes), or protein domains thereof, can charide backbone. Cross-linking chemistries for Such poly also be used as hydrogel monomers. The range of non saccharides are known in the art, see for example Thermo natural (unnatural) amino acids that can be incorporated into Scientific Crosslinking Technical Handbook entitled “Easy such compositions is well known to those skilled in the art molecular bonding crosslinking technology,” (available at (Zhang et al. (2013). Current Opinion in Structural Biology tools.lifetechnologies.com/content/sfs/brochures/1602163 23, pp. 581-587; incorporated by reference in its entirety for Crosslinking-Reagents-Handbook.pdf). all purposes). The biodegradable polymer in one embodi Hyaluronan in one embodiment is used as a hydrogel ment, is used as a co-monomer, i.e., in a mixture of mono monomer (either as a single monomer or as a co-monomer). mers. The biodegradable polymer in one embodiment is a 25 Hyaluronan in one embodiment, is functionalized, for bifunctional monomer. example with acrylate or acrylamide. Hyaluronan is a high The biomonomer, in one embodiment, is functionalized molecular weight GAG composed of disaccharide repeating with acrylamide or acrylate. For example, in one embodi units of N-acetylglucosamine and glucuronic acid linked ment, the polymerizable acrylamide functionalized biomol together through alternating B-1.4 and B-1.3 glycosidic ecule is an acrylamide or acrylate functionalized protein (for 30 bonds. In the human body, hyaluronate is found in several example, an acrylamide functionalized collagen or function Soft connective tissues, including skin, umbilical cord, Syn alized collagen domain), an acrylamide or acrylate function ovial fluid, and vitreous humor. Accordingly, in one embodi alized peptide, or an acrylamide or acrylate functionalized ment, where one or more optical properties of a skin cell, monosaccharide, disaccharide or polysaccharide. umbilical cord cell or vitreous humor cell is desired to be Any monosaccharide, disaccharide or polysaccharide 35 mimicked, in one embodiment, hyaluronan is used as a (functionalized or otherwise) can be used as a hydrogel hydrogel monomer. Methods for fabricating hydrogel par monomer. In one embodiment, an acrylamide or acrylate ticles are described in Xu et al. (2012). Soft Matter. 8, pp. functionalized monosaccharide, disaccharide or polysaccha 3280-3294, the disclosure of which is incorporated herein in ride is used as a polymerizable hydrogel monomer. In one its entirety for all purposes. As described therein, hyaluronan embodiment, a structural polysaccharide is used as a polym 40 can be derivatized with various reactive handles depending erizable hydrogel monomer. In a further embodiment, the on the desired cross-linking chemistry and other monomers structural polysaccharide is an arabinoxylan, cellulose, chi used to form a hydrogel particle. tin or a pectin. In another embodiment, alginic acid (alg In yet other embodiments, chitosan, a linear polysaccha inate) is used as a polymerizable hydrogel monomer. In yet ride composed of randomly distributed B-(1-4)-linked another embodiment, a glycosaminoglycan (GAG) is used 45 D-glucosamine (deacetylated unit) and N-acetyl-D-glu as a polymerizable monomer in the hydrogels provided cosamine (acetylated unit), is used as a hydrogel monomer herein. In a further embodiment, the GAG is chondroitin (either as a single monomer or as a co-monomer). Sulfate, dermatan Sulfate, keratin Sulfate, heparin, heparin Other polysaccharides for use as a hydrogel monomer or sulfate or hyaluronic acid (also referred to in the art as co-monomer include but are not limited to, agar, agarose, hyaluron or hyaluronate) is used as a polymerizable hydro 50 alginic acid, alguronic acid, alpha glucan, amylopectin, gel monomer. The additional range of compatible biomono amylose, arabinoxylan, beta-glucan, callose, capsullan, car mers and their reactive chemistries are known be individuals rageenan polysaccharides (e.g., kappa, iota or lambda class), skilled in the art and follow general chemical reactivity cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolami principles. narin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, An additional range of biocompatible monomers that can 55 ficoll, fructan, fucoidan, galactoglucomannan, galactoman be incorporated are known in the art, see, for example the nan, galactosaminoogalactan, gellan gum, glucan, gluco non-degradable biocompatible monomers disclosed in Shas mannan, glucorunoXylan, glycocalyx, glycogen, hemicellu tri (2003). Current Pharmaceutical Biotechnology 4, pp. lose, homopolysaccharide, hypromellose, icodextrin, inulin, 331-337, incorporated by reference herein in its entirety for kefiran, laminarin, lentinan, levan polysaccharide, lichenin, all purposes. Other monomers are provided in de Moraes 60 mannan, mixed-linkage glucan, paramylon, pectic acid, pec Porto (2012). Polymer Biocompatibility, Polymerization, tin, pentastarch, phytoglycogen, pleuran, polydextrose, Dr. Ailton De Souza Gomes (Ed.), ISBN: 978-953-51-0745 polysaccharide peptide, porphyran, pullulan, Schizophyllan, 3: InTech, DOI: 10.5772/47786; Heller et al. (2010). Journal sinistrin, sizofiran, welan gum, Xanthan gum, Xylan, Xylog of Polymer Science Part A: Polymer Chemistry 49, pp. lucan, Zymosan, or a combination thereof. As described 650-661; Final Report for Biocompatible Materials (2004), 65 throughout, depending on the desired cross-linking chemis The Board of the Biocompatible Materials and the Molecu try and/or additional co-monomers employed in the hydro lar Engineering in Polymer Science programmes, ISBN gel, the polysaccharide can be further functionalized. For US 9,714,897 B2 11 12 example, one or more of the polysaccharides described limited to ethylene glycol dimethacrylate (EGDMA), tetra herein in one embodiment is functionalized with acrylate or ethylene glycol dimethacrylate, and N,N'-15 methylenebi acrylamide. sacrylamide. The range of additional crosslinking chemis In one embodiment, an individual hydrogel particle or a tries which can be used are obvious to those skilled in the art plurality thereof comprises a peptide, protein, a protein 5 and follow general chemical reactivity principles, see for domain, or a combination thereofas a hydrogel monomer or example Thermo Scientific Crosslinking Technical Hand plurality thereof. In a further embodiment, the protein is a book entitled "Easy molecular bonding crosslinking tech structural protein, or a domain thereof, for example, such as nology,” (available at tools.lifetechnologies.com/content/ silk, elastin, titlin or collagen, or a domain thereof. In one sfs/brochures/1602163-Crosslinking-Reagents embodiment, the protein is an extracellular matrix (ECM) 10 Handbook.pdf). component (e.g., collagen, elastin, proteoglycan). In even a In one embodiment, polymerization of a hydrogel is further embodiment, the structural protein is collagen. In yet initiated by a persulfate or an equivalent initiator that a further embodiment, the collagen is collagen type I. catalyzes radical formation. The range of compatible initia collagen type II or collagen type III or a combination tors are known to those skilled in the art and follow general thereof. In another embodiment, the hydrogel monomer 15 chemical reactivity principles, see for example Thermo comprises a proteoglycan. In a further embodiment, the Scientific Crosslinking Technical Handbook entitled “Easy proteoglycan is decorin, biglycan, testican, bikunin, fibro molecular bonding crosslinking technology,” (available at modulin, lumican, or a domain thereof. tools.lifetechnologies.com/content/sfs/brochures/1602163 In another embodiment, an acrylate-functionalized struc Crosslinking-Reagents-Handbook.pdf). The persulfate can tural protein hydrogel monomer is used as a component of be any water-soluble persulfate. Non-limiting examples of the hydrogel provided herein (e.g., an acrylate functional water soluble persulfates are ammonium persulfate and ized protein or protein domain, for example, silk, elastin, alkali metal persulfates. Alkali metals include lithium, titin, collagen, proteoglycan, or a functionalized domain Sodium and potassium. In some embodiments, the persulfate thereof). In a further embodiment, the acrylate functional is ammonium persulfate or potassium persulfate. In a further ized structural protein hydrogel monomer comprises a pro 25 embodiment, polymerization of the hydrogel provided teoglycan, e.g., decorin, biglycan, testican, bikunin, fibro herein is initiated by ammonium persulfate. modulin, lumican, or a domain thereof. Polymerization of a hydrogel can be accelerated by an In one embodiment PEG monomers and oligopeptides accelerant which can catalyze the formation of polymeriza can be that mimic extracellular matrix proteins are used in tion-labile chemical side groups. The range of possible the hydrogels provided herein, for example, with vinyl 30 accelerants is known to those skilled in the art and follow Sulfone-functionalized multiarm PEG, integrin binding pep general chemical reactivity principles see for example tides and bis-cysteine matrix metalloproteinase peptides as Thermo Scientific Crosslinking Technical Handbook described by Lutolf et al. (2003). Proc. Natl. Acad. Sci. entitled "Easy molecular bonding crosslinking technology,” U.S.A. 100, 5413-5418, incorporated by reference in its (available at tools.lifetechnologies.com/content/sfs/bro entirety for all purposes. In this particular embodiment, 35 chures/1602163-Crosslinking-Reagents-Handbook.pdf). hydrogels are formed by a Michael-type addition reaction The accelerant in one embodiment, is a tertiary amine. The between the di-thiolated oligopeptides and vinyl sulfone tertiary amine can be any water-soluble tertiary amine. In groups on the PEG. The range of additional compatible one embodiment, an accelerant is used in the polymerization chemistries that can be incorporated here are obvious to reaction and is N.N.N',N'tetramethylethylenediamine, 3-di those skilled in the art and follow general chemical reactiv 40 methylamino) propionitrile, or N.N.N',N'tetramethylethyl ity principles, see for example Thermo Scientific Crosslink enediamine (TEMED). In another embodiment, an acceler ing Technical Handbook entitled “Easy molecular bonding ant is used in the polymerization reaction and isaZobis crosslinking technology,” (available at tools.lifetechnologi (isobutyronitrile) (AIBN). es.com/content/sfs/brochures/1602163-Crosslinking-Re As discussed above, the hydrogel for use in the compo agents-Handbook.pdf). 45 sitions and methods described herein can include any of the Other bioactive domains in natural proteins can also be monomeric units and crosslinkers as described herein, and in used as a hydrogel monomer or portion thereof. For one aspect, are produced as hydrogel particles by polymer example, a cell-adhesive integrin binding domain, a con izing droplets (see, e.g., FIG. 2). Microfluidic methods of trolled release affinity binding domain or a transglutaminase producing a plurality of droplets, including fluidic and cross-linking domain can be used in the hydrogels provided 50 rigidified droplets, are known to those of ordinary skill in the herein. Details for producing Such hydrogels can be found in art, and described in US Patent Publication No. 2011/ Martino et al. (2009). Biomaterials 30, 1089; Martino et al. 0218123 and U.S. Pat. No. 7,294,503, each incorporated (2011). Sci. Trans. Med. 3, 100ra89: Hu and Messersmith herein by reference in their entireties for all purposes. Such (2003). J. Am. Chem. Soc. 125, 14298, each of which is methods provide for a plurality of droplets containing a first incorporated by reference in its entirety for all purposes. 55 fluid and being substantially surrounded by a second fluid, In one embodiment, recombinant DNA methods are used where the first fluid and the second fluid are substantially to create proteins, designed to gel in response to changes in immiscible (e.g., droplets containing an aqueous-based liq pH or temperature, for example, by the methods described uid being Substantially Surrounded by an oil based liquid). by Petka et al. (1998). Science 281, pp. 389-392, incorpo A plurality of fluidic droplets (e.g., prepared using a rated by reference in its entirety for all purposes. Briefly, the 60 microfluidic device) may be polydisperse (e.g., having a proteins consist of terminal leucine Zipper domains flanking range of different sizes), or in Some cases, the fluidic a water-soluble polyelectrolyte segment. In near-neutral droplets may be monodisperse or Substantially monodis aqueous solutions, coiled-coil aggregates of the terminal perse, e.g., having a homogenous distribution of diameters, domains form a three-dimensional hydrogel polymer net for instance, such that no more than about 10%, about 5%, work. 65 about 3%, about 1%, about 0.03%, or about 0.01% of the Common cross linking agents that can be used to cross droplets have an average diameter greater than about 10%, link the hydrogels provided herein include but are not about 5%, about 3%, about 1%, about 0.03%, or about US 9,714,897 B2 13 14 0.01% of the average diameter. The average diameter of a polymerization, the monomer is insoluble in the continuous population of droplets, as used herein, refers to the arith phase, for example an aqueous monomer Solution in a metic average of the diameters of the droplets. Average continuous oil phase. In Suspension polymerization, polym diameters of the particles can be measured, for example, by erization initiation occurs within the monomer-rich droplets light scattering techniques. Average diameters of hydrogel and with greater than one radical per droplet at any time. The particles in one embodiment, are tailored, for example by monomer phase in one embodiment includes a monomer varying flow rates of the fluid streams of the first and second which can be a bifunctional monomer or a plurality of fluids within the channel(s) of a microfluidic device, or by monomer species (co-monomers, which can be a plurality of varying the volume of the channel(s) of the microfluidic bifunctional monomers. The monomer phase in one embodi device. 10 Accordingly, the disclosure provides population of hydro ment, includes an initiator and/or a crosslinking agent. gel particles comprising a plurality of hydrogel particles, Emulsion polymerization can also be used to form the wherein the population of hydrogel particles is substantially hydrogel particles described herein. In emulsion polymer monodisperse. ization, the monomer has poor solubility in the continuous The term microfluidic refers to a device, apparatus or 15 phase, similar to Suspension polymerization, however, system including at least one fluid channel having a cross polymerization initiation occurs outside the monomer drop sectional dimension of less than 1 mm, and a ratio of length lets (see Elbert (2011). Acta Biomater 7, pp. 31-56, incor to largest cross-sectional dimension perpendicular to the porated by reference herein in its entirety for all purposes). channel of at least about 3:1. A micro fluidic device com In emulsion polymerization embodiments, the initiator prising a micro fluidic channel is especially well Suited to causes chain growth of the monomer (or co-monomers) preparing a plurality of mono disperse droplets. dissolved in the continuous phase or monomer contained in Non-limiting examples of microfluidic systems that may micelles if Surfactants are present. be used with the present invention are disclosed in U.S. In another embodiment, hydrogel particles are formed by Patent Application Publication No. 2006/0163385; U.S. precipitation polymerization, for example as described in Patent Application Publication No. 2005/0172476; U.S. 25 Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by Patent Application Publication No. 2007/000342: Interna reference herein in its entirety for all purposes. Precipitation tional Patent Application Publication No. WO 2006/096571; polymerization is a technique that takes advantage of the U.S. Patent Application Publication No. 2007/0054119; U.S. differences in the solubility of monomer and polymer to Pat. No. 7,776,927; and International Patent Application produce microparticles. Specifically, it is known that larger Publication No. WO 2006/078841, each incorporated herein 30 polymer chains generally have lower solubility than Smaller by reference in their entireties for all purposes. ones. Accordingly, above a specific molecular weight, phase Droplet size is related to microfluidic channel size. The separation may be favored. Precipitation polymerization micro fluidic channel may be of any size, for example, initially begins as solution polymerizations in a single phase, having a largest dimension perpendicular to fluid flow of less homogenous system. Shortly after the start of the polymer than about 5 mm or 2 mm, or less than about 1 mm, or less 35 ization, in one embodiment, a relatively high concentration than about 500 um, less than about 200 um, less than about of polymer chains is present, favoring phase separation by 100 um, less than about 60 lum, less than about 50 um, less nucleation. As polymerization proceeds, the concentration than about 40 um, less than about 30 um, less than about 25 of polymer chains is low and existing particles capture the um, less than about 10 Jum, less than about 3 Lim, less than chains before nucleation of new particles can occur. Thus, about 1 um, less than about 300 nm, less than about 100 nm, 40 nucleation of particles occurs only for a brief period of time less than about 30 nm, or less than about 10 nm. shortly after the start of the reaction, which in one embodi Droplet size can be tuned by adjusting the relative flow ment, results in a narrow size distribution of particles. rates. In some embodiments, drop diameters are equivalent Additional methods include but are not limited to litho to the width of the channel, or within about 10%, 15%, 20%, graphic particle formation (Helgeson et al. (2011). Curr. 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the width 45 Opin. Colloid. Interface Sci. 16, pp. 106-117, incorporated of the channel. by reference herein in its entirety for all purposes) mem The dimensions of a hydrogel particle of the disclosure brane emulsification (e.g., by the micosieve emulsification are substantially similar to the droplet from which it was technology techniques described by Nanomi B.V. (Nether formed. Therefore, in some embodiments, a hydrogel par lands)) and microchannel emulsification (Sugiura et al. ticle has a diameter of less than about 1 um, 2, 5, 10, 15, 20, 50 (2002). Languimir 18, pp. 5708-5712, incorporated by ref 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, erence herein in its entirety) and bulk emulsification (SNF 250, 300, 350, 400, 450, 500, 600, 800, or less than 1000 um Floerger, available at Snf.com.au/downloads/ in diameter. In some embodiments, a hydrogel particle has Emulsion Handbook E.pdf, incorporated by reference a diameter of more than about 1 um, 2, 5, 10, 15, 20, 25, 30. herein in its entirety). 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 55 In one embodiment, hydrogel particles are formed within 350, 400, 450, 500, 600, 800, or greater than 1000 um in a microfluidic device having two oil channels that focus on diameter. In one embodiment, a hydrogel particle has a a central stream of aqueous monomer Solution. In this diameter in the range of 5 um to 100 um. embodiment, droplets form at the interface of the two In some embodiments, a hydrogel particle of the disclo channels and central stream to break off droplets in water Sure is spherical in shape. 60 in-oil emulsion. Once droplets are formed, in one embodi In some embodiments, a hydrogel particle of the disclo ment, they are stabilized prior to polymerization, for Sure does not comprise agarose. example, by adding a surfactant to the oil phase. However, Hydrogel particles in one embodiment, is carried by in another embodiment, droplets are not stabilized prior to Suspension polymerization, which is also referred to in the polymerization. Polymerization of the monomer in one art as pearl, bead or granular polymerization (see Elbert 65 embodiment is triggered by adding an accelerator (e.g., (2011). Acta Biomater 7, pp. 31-56, incorporated by refer N.N.N',N'tetramethylethylenediamine) to one or both of the ence herein in its entirety for all purposes). In Suspension oil channels after initial droplets are formed. US 9,714,897 B2 15 16 The aqueous monomer Solution as provided above can amines, copper catalyzed click chemistry (Sharpless)). The include a single monomer species or a plurality of monomer range of possible embedded molecules which contain com species. The aqueous monomer Solution can include co patible chemistries is understood by those skilled in the art. monomers, a bifunctional monomer or a combination In one embodiment, different subpopulations of hydrogel thereof. In one embodiment, the monomer or plurality of 5 particles are fabricated, each with a different concentration monomers can includes a bifunctional monomer, for of biomolecule. In a further embodiment, the biomolecule is example, one of the monomers described above. As a nucleic acid, a protein, an intracellular ion Such as calcium described below, co-monomers can be used to modulate acid (or other biomolecule of the user's choosing, for forward Scatter or side scatter, for example, by adjusting the example, calcium). In another embodiment, different Sub refractive index of the hydrogel particle. 10 populations of hydrogel particles are fabricated, each with a In one embodiment, the central stream of aqueous mono different concentration of a drug Substance. The drug Sub mer Solution comprises a cross-linker, for example, N,N'- stance in one embodiment is a biomolecule (i.e., a biologic, bisacrylamide. In a further embodiment, the central stream antibody, antibody drug conjugate, protein/enzyme, peptide, of aqueous monomer Solution comprises a cross-linker and non-ribosomal peptide, or related molecule) or a small an accelerator, in addition to the monomer. In yet a further 15 molecule synthetic drug (e.g., Type I/II/III polyketide, non embodiment, the aqueous monomer Solution comprises an ribosomal peptide with bioactive properties, or other small initiator, for example an oxidizing agent Such as ammonium molecule entity as generally classified by those skilled in the persulfate. art). Forward scatter was modulated by adjusting the refractive In this regard, the present invention is particularly useful index of the gel by adding co-monomers allyl acrylate and for determining assay resolution where cells are stained for allyl methacrylate (see also FIGS. 11 and 12). Forward their respective nucleic acid or protein content. In one scatter can also be modulated with side scattering nanopar embodiment, different populations of the hydrogel particles ticles containing Sufficient optical resolution/size/density provided herein are encapsulated with known, differing including, but not limited to, higher density colloidal sus amounts of an intracellular Substance, e.g., nucleic acid or pensions of silica and/or PMMA particles. Side scattering of 25 protein. Individual hydrogel particles are stained for the the droplets was tuned by adding a colloidal Suspension of intracellular Substance and fluorescence is measured via a silica nanoparticles and/or PMMA (poly(methyl methacry cytometric device for the individual hydrogels of the various late)) particles (~100 nm) to the central aqueous phase prior populations. This allows for a generation of a standard curve to polymerization (FIGS. 11 and 12). to establish the sensitivity and dynamic range of the intra In one embodiment, a bead, plurality of beads, biomol 30 cellular assay. Once established, a sample can be run through ecule, or plurality of biomolecules is embedded (encapsu the cytometer to detect target cell(s) if present, and to lated) within the hydrogel particle. An encapsulated bead or quantify the amount of intracellular substance in the respec biomolecule, in one embodiment, is employed to mimic one tive target cell(s). In one embodiment, the embedded sub or more intracellular organelles of a target cell, or a cell after stance is an infectious disease biomarker, for example one of it engulfs a particle. In one embodiment, encapsulating or 35 the infectious disease biomarkers in the Infectious Disease embedding a bead or biomolecule is accomplished at the Biomarker Database (IDBD, see Yang et al. (2008) IDBD: time of hydrogel particle formation. For example, beads can Infectious Disease Biomarker Database. Nucleic Acid Res. be suspended in the appropriate concentration to allow for 36, pp. D455-D460, incorporated by reference in its entirety an average of one bead to be embedded/encapsulated in a for all purposes). In a further embodiment, the infectious single hydrogel particle. The bead Suspension can be 40 disease biomarker is a biomarker of gastrointestinal infec included, for example, within the aqueous solution of mono tion, respiratory infection, neurological infection, urogenital mer. Similarly, a biomolecule or mixture of biomolecules infection, viral infection, hemorrhagic fever, Zoonosis, arbo can be incorporated into the aqueous solution of monomer to virus, antibiotics resistance or bioterrorism. In a further encapsulate the biomolecule or biomolecules. embodiment, the viral infection is an Ebola infection. Alternatively, once a hydrogel particle is formed, for 45 In one embodiment, the methods provided herein are used example by the methods described above, in one embodi to determine the sensitivity and/or dynamic range of a ment, it can be further manipulated, for example, by embed cellular nucleic acid quantification assay. In this embodi ding a bead, plurality of beads, biomolecule or plurality of ment, a sample is interrogated for cell types within the biomolecules within the hydrogel particle. sample (if present), and amount of cellular nucleic acid Accordingly, in one aspect of the invention, a hydrogel 50 within the cell. comprising an embedded Substance is provided. In another embodiment, the present invention provides a In one embodiment, the embedded substance is an embed means for determining the resolution and/or sensitivity of an ded molecule, for example a biomolecule. The biomolecule intracellular protein quantification assay. Hydrogel particles, can be a single species or a plurality of different species. For in one embodiment, encapsulate known amounts of protein, example, a protein, peptide, carbohydrate, nucleic acid or 55 at various concentrations, and Subsequently stained with the combination thereof can be encapsulated within a hydrogel appropriate protein antibody. Fluorescence is measured for particle of the invention. Moreover, different nucleic acid the various particles to determine the sensitivity and/or molecules (e.g., of varying sequences or nucleic acid type dynamic range of the assay. The fluorescence values can such as genomic DNA, messenger RNA or DNA-RNA then be compared to the values obtained from cells in a hybrids) can be encapsulated by the hydrogel particle of the 60 sample, to determine whether a target cell is present and invention. These can be comprised of any protein or nucleic whether it contains the intracellular protein, and the amount acid as both forms of biological material contain labile of the protein. chemical side-groups (or can be modified by commercial In one embodiment, individual hydrogel particles are vendors (e.g., Integrated DNA Technology chemical side tuned to have at least one optical property Substantially group modifications). Such side-groups are compatible with 65 similar to a circulating tumor cell or a fetal cell, present in reaction chemistries commonly found in co-monomer com maternal blood. The individual particles are embedded with positions (e.g. acrylate chemistry, NETS-ester, primary known quantities of a biomolecule of interest. The particles US 9,714,897 B2 17 18 are used to generate a standard curve for a biomolecule RhodolgreenTM carboxylic acid, N.O-bis-(trifluoroacetyl) or detection assay for the particular cell type. Succinimidylester, bis-(4-carboxypiperidinyl) sulfonerhod As provided above, in one aspect of the invention, a amine or di(Succinimidylester), 5-(and-6)carboxynaphtho hydrogel comprising an embedded Substance is provided. In fluorescein, 5-(and-6)carboxynaphthofluorescein Succinim one embodiment, the embedded substance is a bead or idyl ester; 5-carboxyrhodamine 6G hydrochloride: plurality of beads. In one embodiment, a hydrogel particle is 6-carboxyrhodamine6Ghydrochloride, 5-carboxyrhodamine embedded with a single bead. In another embodiment, 6G succinimidyl ester; 6-carboxyrhodamine 6G succinim individual hydrogels the average number of embedded beads idyl ester, 5-(and-6)-carboxyrhodamine(5G succinimidyl in a plurality of hydrogel particles is one. ester, 5-carboxy-2',4',5'7"-tetrabromosulfonefluorescein In the case where a bead or plurality of beads are 10 Succinimidyl esteror bis-(diisopropylethylammonium) salt; embedded into a hydrogel particle, in one embodiment, the 5-carboxytetramethylrhodamine; 6-carboxytetramethylrho optical properties of the bead or plurality of beads are used damine; 5-(and-6)-carboxytetramethylrhodamine; 5-car in combination with the FSC and SSC properties of the boxytetramethylrhodamine succinimidyl ester; 6-carboxyte hydrogel particle for quality control of a flow cytometry tramethylrhodaminesuccinimidyl ester, 5-(and -6)- assay. For example, the embedded bead in one embodiment 15 carboxytetramethylrhodamine Succinimidyl ester, is used as a control to calibrate the flow cytometer system, 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine Succin including the laser Source, optics, and stream flow. In imidyl ester; 6-carboxy-Xrhodamine succinimidyl ester; another embodiment, the embedded bead is used as a means 5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester; 5-car for quantitating the amount of fluorescence in a sample, e.g., boxy-X-rhodamine triethylammonium salt; LissamineTM a particular cell. In this regard, embedded beads of various rhodamine B sulfonyl chloride; malachite green; isothiocya intensities can be used to generate a standard curve of nate; NANOGOLDR) mono(sulfosuccinimidyl ester); fluorescence to determine whether a cell expresses a certain QSYR 21 carboxylic acid or succinimidyl ester; QSYR 7 marker and at what level of expression. carboxylic acid or succinimidyl ester; Rhodamine RedTM-X In one embodiment, a bead with the diameter of about 1 Succinimidyl ester, 6-(tetramethylrhodamine-5-(and-6)-car um to about 3 um, about 2 um to about 4 um or about 3 um 25 boxamido) hexanoic acid; Succinimidyl ester, tetramethyl to about 7 um is embedded in a hydrogel provided herein. rhodamine-5-isothiocyanate; tetramethylrhodamine-6-isoth For example, in one embodiment, the bead has a diameter of iocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; about 3 um to about 3.5 um. In a further embodiment, the Texas Red R) sulfonyl: Texas Red R) sulfonyl chloride; Texas bead is a fluorescent bead. In another embodiment, the bead Red R-X STP ester or sodium salt, Texas Red R-X succin has a diameter of about 1 um to about 2.5 um or about 1.5 30 imidyl ester, Texas Red R-X succinimidyl ester; and X-rho um to about 3 um. In a further embodiment, the bead is a damine-5-(and-6) isothiocyanate, BODIPY(R) dyes commer fluorescent bead and can be stained either internally or at its cially available from Invitrogen, including, but not limited to surface. In even a further embodiment, the fluorescent bead BODIPYR FL; BODIPYR TMR STP ester; BODIPY(R) is stained internally. Without wishing to be bound by theory, TR-X STP ester BODIPYR) 630/650-X STPester; it is thought that internal staining insulates the fluorophores 35 BODIPYR 650/665-X STP ester; 6-dibromo-4,4-difluoro from environmental interactions that could cause variable 5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionic fluorescence output. acid Succinimidyl ester, 4.4-difluoro-4-bora-3a,4a-diaza-S- As provided above, in one embodiment, the embedded indacene-3,5-dipropionic acid; 4.4-difluoro-5,7-dimethyl-4- bead is a fluorescence bead and in a further embodiment, the bora-3a,4a-diaza-S-indacene-3-pentanoicacid; 4.4-difluoro fluorescent bead is stained internally. It is within the skill in 40 5,7-dimethyl-4-bora3a.4a-diaza-S-indacene-3-pentanoicacid the art to select the appropriate fluorophore for use in succinimidyl ester; 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a conjunction with an embedded bead. In one embodiment, diaza-S-indacene-3propionicacid; 4,4-difluoro-5,7-dim the bead is derivatized with one or more of the following ethyl-4-bora-3a,4adiaza-S-indacene-3-propionicacid Succin fluorescent dyes: 6-carboxy-4',5'-dichloro-2',7-dimethoxy imidyl ester; 4.4difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza fluorescein Succinimidylester, 5-(and-6)-carboxyeosin; 45 S-indacene-3propionic acid; Sulfo Succinimidyl ester or 5-carboxyfluorescein; 6 carboxyfluorescein; 5-(and-6)-car sodium salt; 6-((4.4-difluoro-5,7-dimethyl-4-bora-3a,4a-di boxyfluorescein; S-carboxyfluorescein-bis-(5-car aza-S-indacene-3propionyl)amino)hexanoicacid: 6-((4.4-di boxymethoxy-2-nitrobenzyl)ether.-alanine-carboxamide, or fluoro-5, 7 dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pro Succinimidyl ester, 5-carboxy fluorescein Succinimidyl pionyl)amino)hexanoic acid or Succinimidyl ester, N-(4.4- ester; 6-carboxyfluorescein succinimidyl ester, 5-(and-6)- 50 difluoro 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotri propionyl) cysteic acid. Succinimidyl ester or azinyl)amino fluorescein; 2, 7-difluorofluorescein; eosin triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4- 5-isothiocyanate; erythrosin5-isothiocyanate; methoxyphenyl)-4-bora3a,4a4,4-difluoro-5,7-diphenyl-4- 6-(fluorescein-5-carboxamido) hexanoic acid or Succinim bora-3a,4a-diaza-Sindacene-3-propionicacid; 4.4-difluoro-5, idyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic 55 7-diphenyl-4-bora3a, 4a-diaza-S-indacene-3-propionicacid acid or succinimidylester; fluorescein-S-EX succinimidyl Succinimidyl ester, 4.4-difluoro-5-phenyl-4-bora-3a,4a-di ester; fluorescein-5-isothiocyanate; fluorescein-6-isothio aza-S-indacene-3-propionic acid; Succinimidyl ester, 6-((4. cyanate; OregonGreen R. 488 carboxylic acid, or succinim 4-difluoro-5-phenyl-4 bora-3a,4a-diaza-S-indacene-3-pro idyl ester; Oregon Green(R) 488 isothiocyanate; Oregon pionyl)amino) hexanoicacid or Succinimidyl ester, 4.4- Green R. 488-X succinimidyl ester; Oregon Green(R) 500 60 difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s- carboxylic acid; Oregon Green R. 500 carboxylic acid, suc indacene-3-propionicacid Succinimidyl ester, 4.4-difluoro cinimidylester or triethylammonium salt; Oregon Green(R) 5-(2-pyrrolyl)-4-bora-3a,4a-diaza-S-indacene-3-propionic 514 carboxylic acid; Oregon Green R 514 carboxylic acid or acid succinimidyl ester; 6-(((4.4-difluoro-5-(2-pyrrolyl)-4- succinimidyl ester; RhodamineGreenTM carboxylic acid, bora-3a,4a-diaza-S-indacene-3-yl)styryloxy)acetyl)amino succinimidyl ester or hydrochloride; Rhodamine GreenTM 65 hexanoicacid or Succinimidyl ester, 4.4-difluoro-5-styryl-4- carboxylic acid, trifluoroacetamide or Succinimidylester, bora-3a,4a-diaza-S-indacene-3-propionic acid; 4. 4-difluoro Rhodamine GreenTM-X succinimidyl ester or hydrochloride: 5-styryl-4-bora-3a,4a-diaza-Sindacene-3-propionic acid; US 9,714,897 B2 19 20 succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora acid; Alexa Fluor R. 488 carboxylic acid; Alexa Fluor R. 532 3a,4adiaza-S-indacene-8-propionicacid; 4.4-difluoro-1,3,5, carboxylic acid; Alexa Fluorr 546 carboxylic acid; Alexa 7-tetramethyl-4bora-3a,4a-diaza-Sindacene-8-propionic Fluor R. 555 carboxylic acid; Alexa Fluor R. 568 carboxylic acid succinimidyl ester; 4.4-difluoro-5-(2-thienyl)-4-bora acid; Alexa Fluor R 594 carboxylic acid; Alexa Fluor R 633 3a,4a-diaza-sindacene-3-propionic acid succinimidyl ester; 5 carboxylic acid; Alexa Fluorr) 647 carboxylic acid; Alexa 6-(((4-(4, 4-difluoro-5-(2-thienyl)-4-bora-3a,4adiazas-in- Fluorr 660 carboxylic acid; and Alexa Fluor R 680 carbox dacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or Succin- ylic acid, cyanine dyes commercially available from Amer imidyl ester; and 6H(4,4-difluoro-5-(2-thienyl)-4-bora-3a, sham-Pharmacia Biotech, including, but not limited to Cy3 4a-diaza-S-indacene-3-yl)styryloxy)acetyl) aminohexanoic NHS ester; Cy5 NHS ester; Cy5.5 NHSester; and Cy7 NHS acid or Succinimidyl ester, Alexa fluor dyes commercially 10 ester. available from Invitrogen, including but not limited to Alexa Other Fluorophores amenable for use with the present Fluor R 350 carboxylic acid; Alexa Fluor R. 430 carboxylic invention are provided in Table 2 below. TABLE 2

Exci- Emis D NAME Alternate Names tation sion Vendorf Source ACS CASii SAC148 6-carboxyfluorescein 492 518 PubChem 3301-79-9 SAC1 6-JOE 52O 550 LifeTechnologies 828SS-40-1 SAC2 7-AAD 545 647 LifeTechnologies 7240-37-1 SAC3 Acridine Orange 503 525 LifeTechnologies 65-61-2 SAC4 Alexa Fluor 350 AF350; 2H-1-Benzopyran-6-sulfonic acid, 343 442 LifeTechnologies 244636-14-4 7-amino-3-2-(2,5-dioxo-1- pyrrollidinyl)oxy-2-oxoethyl-4-methyl-2- oxo-: 200554-19-4 SAC6 Alexa Fluor 405 AF405; C46H69N5O15S3 401 425 LifeTechnologies 791637-08-6 SAC7 Alexa Fluor 430 AF430; C32H42F3N3O9S 433 541 LifeTechnologies 467233-94-9 SAC8 Alexa Fluor 488 AF488; C25H15Li2N3O13S2 496 519 LifeTechnologies 247144-99-6 SAC9 Alexa Fluor 500 AF500; CASH 798557-08-1 503 525 LifeTechnologies 798.557-08-1 SAC10 Alexa Fluor 514 AF514; C31H27N3O13S2 517 542 LifeTechnologies 798.557-07-0 SAC11 Alexa Fluor 532 AF532; 1 H-Pyrano3,2-f: 5,6-foliindole- 532 553 LifeTechnologies 2221.59-92-4 10,12-disulfonic acid, 5-4-(2,5-dioxo-1- pyrrollidinyl)oxycarbonylphenyl-2,3,7,8- tetrahydro-2,3,3,7,7,8-hexamethyl-, 271795-14-3 SAC13 Alexa. Fluor 546 AF546; CSOH62C3N5O14S3 556 573 LifeTechnologies 247145-23-9 SAC14 Alexa Fluor 555 AF555 555 565 LifeTechnologies 644990-77-2 SAC15 Alexa Fluor 568 AFS68 578 603 LifeTechnologies 247145-38-6 SAC16 Alexa Fluor 594 AFS94 590 617 LifeTechnologies 247145-86-4 SAC17 Alexa Fluor 610 AF610; C58H77C3N6O14S3 612 628 LifeTechnologies 90OS28-62-3 SAC18 Alexa Fluor 633 AF633 632 647 LifeTechnologies 477780-06-6 SAC19 Alexa Fluor 635 AF635 633 647 LifeTechnologies 94.58SO-82-8 SAC2O Alexa Fluor 647 AF647 6SO 665 LifeTechnologies 400051-23-2 SAC21 Alexa Fluor 660 AF660 663 690 LifeTechnologies 422.309-89-5 SAC22 Alexa Fluor 680 AF68O 679 702 LifeTechnologies 422.309-67-9 SAC23 Alexa Fluor 700 AF700 702 723 LifeTechnologies 697795-05-4 SAC24 Alexa Fluor 750 AF750 749 775 LifeTechnologies 697795-06-5 SAC25 Alexa Fluor 790 AF790 784 814 LifeTechnologies 950891-33-5 SAC26 AMCA 346 448 SantaCruzBiotech 106562-32-7 SAC27 AmCyan 457 489 BDBioscences 1216872-44-4 SAC28 APC Allophycocyanin 6SO 660 Sigma Aldrich No names found SAC29 APC-Alexa Fluor 680 APC-AF68O 655 704 LifeTechnologies No names found SAC30 APC-Alexa Fluor 700 APC-AF700 655 718 LifeTechnologies No names found SAC31 APC-Alexa Fluor 750 APC-AF7SO 6SO 775 LifeTechnologies No names found SAC32 APC-Cy5.5 Allophycocyanin-Cy5.5 6SO 695 LifeTechnologies No names found SAC33 APC-Cy7 Allophycocyanin-Cy7 6SO 767 LifeTechnologies No names found SAC34 APC-eFluor 750 eFlorf SOAPC 6SO 750 eBioscience No names found SAC3S APC-eFluor 780 eFluor780APC 6SO 780 eBioscience 472O56-77-1 SAC36 APC-H7 H7APC 6SO 765 BDBioscences 366OOO-62-5 SAC37 APC-ViO770 ViO77OAPC 652 775 Miltenyl Biotech No names found SAC38 Atto488 5O1 523 ATTO-TEC 923585-42-6 SAC39 BIOTIN O O PubChem S8-85-S SAC4O BODIPY FL 502 511 SantaCruzBiotech 65599-63-3 SAC41 BODIPY R6G 44-difluoro-5-phenyl-4-bora-3.a4a-diaza- 527 547 LifeTechnologies 33S193-70-9 S-indacene-3-propionic acid, Succinimidyl ester; C22H18BF2N3O4. SAC43 Brilliant Violet 421 BV421 406 423 Biolegend 428441-68-2 SAC44 Brilliant Violet 510 BV510 40S 510 Biolegend No names found SAC45 Brilliant Violet 570 BV570 407 571 Biolegend 428441-76-2 SAC46 Brilliant Violet 605 BV605 407 603 Biolegend 63.2128-60-9 SAC47 Brilliant Violet 612 BV612 O O Biolegend 428441-91-1 SAC48 Brilliant Violet 650 BV6SO 407 647 Biolegend No names found SAC49 Brilliant Violet 711 BV711 40S 711 Biolegend No names found SACSO Brilliant Violet 785 BV785 40S 786 Biolegend 613592-44-1 SACS3 Calcein CASii: 1461-15-O 493 514 LifeTechnologies 461-15-O SACS1 Calceiin AM 496 517 PubChem 48504-34-1 SACS2 Calceiin Blue AM 360 445 PubChem 684.82-84-6 SACS4 Calcein Violet AM 400 452 LifeTechnologies No names found US 9,714,897 B2 21 22 TABLE 2-continued

Exci- Emis D NAME Alternate Names tation sion Vendorf Source ACS CASii SACSS Calcium Sensor Dye eFluor 490 514 eBioscience No names found S1.4 SACS6 Cascade Blue 4O1 420 PubChem 1325-87-7 SACS7 Cascade Yellow 400 550 Synchem UG & Co. 220930-95-0 KG SACS8 Cell Proliferation Dye 40S 445 eBioscience No names found eFluor 450 SACS9 Cell Proliferation Dye 652 672 eBioscience No names found eFluor 670 SAC6O CelTrace Violet Cell 392 455 LifeTechnologies No names found Proliferation SAC61 CelVue Claret 655 657 Sigma Aldrich O42142-46-0 SAC62 CFSE 492 525 SantaCruzBiotech SO347-59-4 SAC63 CPC Olcresolphthalein complexone 488 660 Chemical Book 2411-89-4 SAC65 Cy2 492 507 GElifeSciences O2185-03-5 SAC66 Cy3 552 566 GElifeSciences 46368-16-3 SAC67 Cy3.5 581 598 GElifeSciences 89767-4S-1 SAC68 Cy5 633 670 GElifeSciences 4.4377-OS-9 SAC69 Cy5.5 677 695 GElifeSciences 21O892-23-2 SAC70 Cy7 743 767 GElifeSciences 69799-14-8 SAC71 Cychrome 565 667 BDBioscences 245670-67-1 SAC73 CyOUANT DNA 502 522 LifeTechnologies No names found SAC74 CyTRAK Orange 1,5-bis(2-(di-methylamino) S1.4 609 Abcam 195771-25-5 ethylamino-4,8-dihydroxyanthracene- (eBioscience) 9,10-dione SAC76 DAPI 358 462 PubChem 47165-04-8 SAC77 DCFEH 505 525 Sigma Aldrich O6070-31-9 SAC79 DiA DiA: 4-Di-16-ASP (4-(4- 455 586 LifeTechnologies 371114-38-4 (Dilhexadecylamino)styryl)-N- Methylpyridinium Iodide); C46H79IN2 SAC81 DD DiD' solid; DiIC18(5) solid (1,1'- 647 669 LifeTechnologies 27274-91-3 Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine, 4 Chlorobenzenesulfonate Salt); C67H103CN2O3S ISAC84 Di DiI Stain (1,1'-Dioctadecyl-3,3,3',3'- 550 568 LifeTechnologies 41085-99-8 Tetramethylindocarbocyanine Perchlorate (DiI: DiIC18(3))); C59H97CIN2O4; 3H indolium, 2-(3-(1,3-dihydro-3,3-dimethyl -octadecyl-2H-indol-2-ylidene)-1- propenyl)-3,3-dimethyl-1-octadecyl-, perchlorate? ISAC88 DO DiO': DiOC18(3) (3,3'- 489 506 LifeTechnologies 3421 S-57-1 Dioctadecyloxacarbocyanine Perchlorate); C53H85CIN2O6; Benzoxazolium, 3-octadecyl-2-3-(3- octadecyl-2(3H)-benzoxazolylidene)-1- propenyl-, perchlorate SAC92 DiR DiR': DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'- 750 781 LifeTechnologies OOO68-60-8 Tetramethylindotricarbocyanine Iodide); C63H101IN2 SAC95 DRAQ5 645 683 CellSignallingTech 254098-36-7 SAC96 DRAQ7 599 694 CellSignallingTech 1533453-55-2 SAC97 DSRED 532 595 Contech 4698.63-23-8 SAC98 dsRed2-RFP 555 582 Contech No names found SAC99 DYS47 547 Dyomics 557 574 Dynomics 9471-38-67-2 SAC1 OO DY634 634 Dyomics 635 658 Dynomics 18901O-49-8 SAC101 DY647 647 Dyomics 6SO 665 Dynomics 89.0317-39-2 SAC1 O2 DyLight 350 DL3SO 353 432 PierceNet 43.6849-83-0 SAC103 DyLight 405 DL405 400 420 PierceNet OS1927-09-3 SAC104 DyLight 488 DL488 493 518 PierceNet OS1927-12-8 SAC1 OS DyLight 549 DLS49 S62 576 JacksonImmunoRes 1051927-13-9 SAC106 DyLight 550 DL550 S62 576 PierceNet 340586-78-8 SAC107 DyLight 594 DLS94 593 618 PierceNet 268612-OO-5 SAC108 DyLight 633 DL633 638 658 PierceNet OS1927-14-0 SAC109 DyLight 649 DL649 654 670 JacksonImmunoRes 1051927-15-1 SAC110 DyLight 650 DL6SO 652 672 PierceNet 364214-13-0 SAC111 DyLight 680 DL68O 682 712 PierceNet OS1927-24-2 SAC112 DyLight 800 DL800 777 794 PierceNet OS1927-23-1 SAC113 EB Ethidium Bromide 523 604 Sigma Aldrich 239-45-8 SAC114 ECD 563 613 LifeTechnologies 88475-75-6 SAC116 ECFP enhanced cyan fluorescent protein 435 477 MyBiosource No names found SAC118 EdU EdU(5-ethynyl-2\u2O32-deoxyuridine); O O LifeTechnologies 61135-33-9 C11H12N2OS SAC12O EdU Alexa Fluor 488 496 516 LifeTechnologies No names found SAC121 EdU Alexa Fluor 647 6SO 665 LifeTechnologies No names found SAC122 EdU Pacific Blue 40S 455 LifeTechnologies No names found US 9,714,897 B2 23 24 TABLE 2-continued

Exci Emis Alternate Names tation Sion Vendorf Source ACS CASii

SAC 23 uor 450 400 450 eBioscience 1592653-87-6 SAC 24 uor 450 Fixable Viability 400 450 eBioscience No names foun

SAC 25 350 490 eBioscience No names Oll SAC 26 uor 506 Fixable Viability 420 SO6 e Bioscience No names Oll e SAC 27 uor 525 350 525 Bioscience No names Oll SAC 28 uor 565 350 565 Bioscience No names foun SAC 29 uor 585 350 604 Bioscience No names foun SAC 30 uor 605 350 60S Bioscience 1248429-27-7 SAC 31 uor 615 590 622 Bioscience No names foun SAC 32 uor 625 350 625 Bioscience No names foun SAC 33 uor 650 350 6SO Bioscience No names foun SAC 34 uor 660 633 658 Bioscience 1634649-1 6-3 SAC 35 uor 670 O O Bioscience 1437243-07-6 SAC 36 uor 700 350 700 Bioscience No names foun SAC 37 uor 710 350 710 Bioscience No names foun SAC 38 uor 780 Fixable Viability 755 78O Bioscience No names foun Dye SAC 39 EGFP enhanced green fluorescent protein 480 510 MyBiosource No names Oll SAC 41 Emerald 300 289 530 LifeTechnologies No names Oll SAC 42 Eosin 525 S46 Sigma Aldrich 1737.2-87-1 SAC 43 Ethidium Homodimer-1 528 617 Sigma Aldrich 61926-22-5 SAC Ethidium Monoazide EMA 510 590 Sigma Aldrich 58880-05-0 SAC 45 EYFP enhanced yellow fluorescent protein 515 528 MyBiosource No names Oll SAC 47 FAM 492 518 PubChem 76823-03-5 SAC 49 FITC Fluorescein 500 520 PubChem 27072-45-3 SAC 53 Fluo-3 C51 H5OCI2N2O23; Glycine, N-(4-6- SO6 526 LifeTechnologies 123632-39-3 (acetyloxy)methoxy-2,7-dichloro-3-oxo 3H-xanthen-9-yl)-2-[2-(2-bis(2- (acetyloxy)methoxy-2-oxyethylamino-5- methylphenoxyethoxyphenyl-N-(2- (acetyloxy)methoxy-2-oxyethyl-, (acetyloxy)methyl ester? ISAC15S Fluo-4 C51 H5OF2N2O23; Glycine, N-(4-6- 494 S16 LifeTechnologies 273221-59-3 (acetyloxy)methoxy-2,7-difluoro-3-oxo 3H-xanthen-9-yl)-2-[2-(2-bis(2- (acetyloxy)methoxy-2-oxoethylamino 5-methylphenoxyethoxyphenyl-N-(2- (acetyloxy)methoxy-2-oxoethyl-, (acetyloxy)methyl ester? SAC 52 FLMA Fluorescein-5-maleimide 495 520 PierceNet 7S3SO-46-8 SAC 57 Fluoro-Emerald Dextran, Fluorescein, 10,000 MW, 495 523 LifeTechnologies 194369-11-4 Anionic, Lysine Fixable SAC 59 Fura Red LifeTechnologies 149732-62-7 SAC 62 Fura Fura-2 LeakRes (AM) 325 510 Sigma Aldrich 172890-84-5 SAC 64 FxCycle Far Red 640 658 LifeTechnologies No names found SAC 65 FxCycle Violet C16H17CI2N5; 1 H-Indole-6- 358 462 LifeTechnologies 28718-90-3 carboximidamide, 2-4- (aminoiminomethyl)phenyl-, dihydrochloride? SAC 67 GFP green fluorescent protein 488 515 MyBiosource No names found SAC 69 GFP Violet Excited 398 515 MyBiosource No names found SAC 70 GFP-Vex1 398 515 MyBiosource No names found SAC 71 HiLyte Fluor 488 5O1 527 Anaspec 1051927-29-7 SAC 72 HiLyte Fluor 555 550 566 Anaspec 1051927-30-0 SAC 73 HiLyte Fluor 647 649 674 Anaspec 925693-87-4 SAC 74 HiLyte Fluor 680 Anaspec 1051927-34-4 SAC 75 HiLyte Fluor 750 754 778 Anaspec 1051927-32-2 SAC 76 Hoechst 33258 345 455 Sigma Aldrich 23491-45-4 SAC 77 Hoechst 33342 bishBenzimide H 33342 trihydrochloride 343 455 Sigma Aldrich 23491-52-3 SAC 79 Hydroxycoumarin C10H6O5; 7-hydroxycoumarin-3- 360 450 LifeTechnologies 43070-85-S carboxylic acid; 2H-1-Benzopyran-3- carboxylic acid, 7-hydroxy-2-oxo-f: 4 chloromethyl-7-hydroxycoumarin SAC 83 Indo-1 Indo-1 AM Calcium Sensor Dye; 347 480 LifeTechnologies 96.314-96-4 C47H51N3O22; 1 H-Indole-6-carboxylic acid, 2-4-bis(2-(acetyloxy)methoxy-2- oxoethylamino-3-2-2-bis(2- (acetyloxy)methoxy-2-oxoetylamino-5- methylphenoxyethoxyphenyl-, (acetyloxy)methyl ester? ISAC187 JC-1 5,5,6,6'-tetrachloro-1,1,3,3'- 593 595 LifeTechnologies tetraethylbenzimidazolylcarbocyanine iodide: C25H27Cl4IN4 ISAC189 Krome Orange 398 530 Beckman Coulter 1558O3S-65-6 ISAC190 Leadmium 490 520 LifeTechnologies No names found US 9,714,897 B2 25 26 TABLE 2-continued

Exci Emis D NAME Alternate Names tation Sion Vendorf Source ACS CASii SAC191 LIVE/DEAD Fixable Aqua Aqua LIVE/DEAD 367 526 LifeTechnologies No names found Dead Cell Stain SAC193 LIVE DEAD Fixable Blue Blue LIVE DEAD 343 442 LifeTechnologies No names found Dead Cell Stain SAC195 LIVE DEAD Fixable Far Red 670 LifeTechnologies No names found Dead Cell Stain SAC196 LIVE DEAD Fixable Green Green LIVE DEAD 498 525 LifeTechnologies No names found Dead Cell Stain SAC198 LIVE DEAD Fixable Near-IR 752 776 LifeTechnologies No names found Dead Cell Stain SAC199 LIVE DEAD Fixable Red 594 612 LifeTechnologies No names found Dead Cell Stain SAC2OO LIVE DEAD Fixable Violet Violet LIVE DEAD 403 455 LifeTechnologies No names found Dead Cell Stain SAC2O2 LIVE DEAD Fixable Yellow Yellow LIVE DEAD 551 LifeTechnologies No names found Dead Cell Stain Lucifer Yellow C13H9Li2NSO9S2; 1 H 428 544 LifeTechnologies 82446-52-4 Benz deisoquinoline-5,8-disulfonic acid, 6-amino-2-(hydrazinocarbonyl)amino 2,3-dihydro-1,3-dioxo-, dilithium salt SAC2O6 Magnesium Green C33H17C12K5N2O13; Glycine, N-(2- 507 531 LifeTechnologies 17OS 16-41-3 (carboxymethoxy)-4-(2',7'-dichloro-3',6'- dihydroxy-3-oxospiro isobenzofuran 1(3H).9-9Hxanthen-5- yl)carbonylaminophenyl-N- (carboxymethyl)-, pentapotassium salt ISAC2O8 Marina Blue C16H11 F2NO7: 2,5-Pyrrollidinedione, 1 364 461 LifeTechnologies 215868-23-8 (6,8-difluoro-7-hydroxy-4-methyl-2-oxo 2H-1-benzopyran-3-yl)acetyl)oxy-f: ISAC210 mBanana S4O 553 Clontech 1114839-40-5 ISAC211 mCherry 587 610 Clontech 1628764-31-7 ISAC212 mCitrine S16 529 Not Commercialized 1357.606-54-2 ISAC213 MethylCoumarin AMCA-X, SE (6-((7-Amino-4- 360 448 LifeTechnologies 1333-47-7 Methylcoumarin-3-Acetyl)amino)Hexanoic Acid, Succinimidyl Ester); C22H25N3O7 ISAC216 MitoTracker Green C34H28Cl5N3O: Benzoxazolium, 2-3- 490 512 LifeTechnologies 1304563-13-0 5,6-dichloro-1,3-bis(4- (chloromethyl)phenyl)methyl-1,3-dihydro 2H-benzimidazol-2-ylidene-1-propenyl 3-methyl-, chloride? SAC218 MitoTracker Orange 550 575 LifeTechnologies No names found SAC219 MitoTracker Red 578 598 LifeTechnologies No names found SAC220 mOrange S48 S62 Clontech 1114839-60-9 SAC221 mPlum 590 649 Clontech 139982O-93-9 SAC222 mRaspberry 597 624 1452799-41-5 SAC223 RFP1 S84 607 SAC224 mStrawberry 574 596 1114834-99-9 SAC225 Na-Green Sodium Green TM, SO6 532 LifeTechnologies 195244-55-4 tetra (tetramethylammonium) salt; C84H10OC4N8O19 SAC228 Nile Red C20H18N2O2; SH 559 637 LifeTechnologies 7385-67-3 Benzo(\u03B1phenoxazin-5-one, 9 (diethylamino)-f SAC230 Oregon Green 491 519 LifeTechnologies 195136-58-4 SAC232 Oregon Green 488-X, 500 525 LifeTechnologies 890416-18-9 Succinimidyl ester SAC233 Oregon Green 514 Oregon Green (R) 514 carboxylic acid, 510 532 LifeTechnologies 198139-53-6 succinimidyl ester: C26H12F5NO9S SAC235 Pacific Blue PacBlue: Pacific Blue TMsuccinimidyl 40S 455 LifeTechnologies 215868-31-8 ester: C14H7F2NO7 SAC236 Pacific Blue succinimidyl 40S 455 LifeTechnologies 215868-33-0 ester SAC237 Pacific Orange PacOrange 403 551 LifeTechnologies 1122414-42-9 SAC240 PE-Alexa Fluor 610 RPE-AF610 563 628 Li echnologies No names found SAC241 PE-Alexa Fluor 647 RPE-AF647 567 669 LifeTechnologies No names found SAC242 PE-Alexa Fluor 680 RPE-AF68O 570 702 LifeTechnologies No names found SAC243 PE-Alexa Fluor 700 RPE-AF700 563 720 LifeTechnologies No names found SAC244 PE-Alexa Fluor 750 RPE-AF7SO 570 776 AbD Serotec No names found SAC245 PE-CF594 PE-Dazzle 594 S64 612 BDBioscences 1613592-67-8 SAC72 PE-Cy5 565 667 BDBioscences 1448849-77-1 SAC248 PE-Cy5.5 563 695 AbD Serotec No names found SAC249 PE-Cy7 563 760 AbD Serotec 1429496-42-3 SAC2SO PE-DY590 563 599 LSBio No names found SAC251 PE-DY647 563 672 LSBio No names found SAC2S2 PerCP 490 675 AbD Serotec 422551-33-5 SAC2S3 PerCP-Cy5.5 488 695 AbD Serotec 1474O26-81-7 SAC254 PerCP-eFor 710 488 710 eBioscience 1353683-31-4 US 9,714,897 B2 27 28 TABLE 2-continued

Exci Emis D NAME Alternate Names tation Sion Vendorf Source ACS CASii SAC115 PE-Texas Red 563 613 LifeTechnologies No names found SAC256 PE-Vio770 565 775 Miltenyl Biotech No names found SAC257 pHrodo pHrodo TM Red, succinimidyl ester S60 S86 LifeTechnologies No names found (pHrodo TM Red, SE); pHrodo TM Green STP Ester SAC260 pHrodo Green STP Ester S60 S86 LifeTechnologies No names found SAC258 pHrodo Red, succinimidyl S60 S86 LifeTechnologies No names found ester SAC261 Phycocyanin 617 646 Sigma Aldrich 11016-15-2 SAC262 Pico Green Quant-iT TM Pico Green (R) dsDNA Reagent 502 522 LifeTechnologies 177571-06-1 SAC264 PKH2 Green Fluorescent Cell Linker 490 SO4 Sigma Aldrich 145687-07-6 SAC266 PKH26 PKH26 Red Fluorescent Cell Linker 551 567 Sigma Aldrich 154214-SS-8 SAC268 PKH67 Green Fluorescent Cell Linker 490 SO4 Sigma Aldrich 257 277-27-3 SAC270 POPO-1 C41 H54I4N6O2; Benzoxazolium, 2,2'- 433 457 LifeTechnologies 169454-15-3 1,3-propanediylbis(dimethyliminio)-3,1- propanediyl-1(4H)-pyridinyl-4- ylidenemethylidynebis3-methyl-, tetraiodide? SAC272 PO-PRO-1 C20H27I2N3O: Benzoxazolium, 3 435 457 LifeTechnologies 1571.99-56-9 methyl-2-1-3- (trimethylammonio)propyl-4(1H)- pyridinylidenelmethyl-, diiodide?: SAC274 Propidium Iodide C27H34I2N4; Phenanthridinium, 3,8- 350 617 LifeTechnologies 2S535-16-4 diamino-5-3- (diethylmethylammonio)propyl-6-phenyl-, diiodide SAC276 PURE Not Commercialized No names found SAC277 Pyronin Y 547 S60 Sigma Aldrich 92-32-O SAC278 Qdot 525 350 525 LifeTechnologies 885332-45-6 SAC279 Qdot 545 350 545 LifeTechnologies 948.906-89-6 SAC28O Qdot 565 350 565 Li echnologies 8S9509-02-7 SAC281 Qdot 585 350 585 Li Technologies 885332-46-7 SAC282 Qdot 605 350 60S Li Technologies 8498.13-89-4 SAC283 Qdot 625 350 625 Li technologies 1144512-19-5 SAC284 Qdot 655 350 655 Li Technologies 674287-64-O SAC28S Qdot 705 350 705 Li Technologies 885332-47-8 SAC286 Qdot 800 350 800 Li Technologies 885332-SO-3 SAC287 RD1 R-Phycoerythrin 563 578 Li Technologies 1376573-14-6 SAC295 Rhodamine 550 570 Li Technologies No names found SAC290 Rho. 110 Rhodamine 110 497 520 Li Technologies 13558-31-1 SAC293 Rho. 123 Rhodamine 123 507 529 Li Technologies 626.69-70-9 SAC296 Rhodamine Green Rhodamine Green TM carboxylic acid, 505 527 Li echnologies 1892OO-71-3 Succinimidyl ester, hydrochloride; C25H18CN3O7 SAC297 Rhodamine Green carboxylic acid, Succinimidyl ester, hydrochloride 505 527 LifeTechnologies 2S4732-34-8 SAC298 Rhodamine Red 573 591 LifeTechnologies 997S2-92-8 SAC299 Rhodamine Red-X Rhodamine Red TM-X, succinimidyl ester; 570 576 LifeTechnologies 178623-12-6 C37H44N4O1 OS2 SAC300 Rhodamine Red-X, 570 576 LifeTechnologies 178623-13-7 Succinimidyl ester SAC301 RiboFlavin 266 531 Sigma Aldrich 83-88-5 SAC239 R-Phycoerythrin PE 563 578 LifeTechnologies 11016-17-4 SAC303 SNARF-1 carboxylic acid, acetate, succinimidyl ester S49 S86 LifeTechnologies No names found SAC3O2 SNARF-1 pH 6 SNARF (R)-1 carboxylic acid, acetate, S49 S86 LifeTechnologies No names found succinimidyl ester; C33H24N2O9 SAC304 SNARF-1 pH 9 576 640 LifeTechnologies No names found SAC3OS Spectral Re SO6 665 MyBiosource No names found SAC306 SureLight P 545 667 Abcam (Columbia No names found Biosciences) SAC307 SureLight P3 614 662 Abcam 1365659-06-8 SAC3O8 SureLight PBXL-3 614 662 Abcam No names found SAC309 SYBR Green 498 522 Sigma Aldrich 217087-73-5 SAC310 SYTO 11 SO6 526 LifeTechnologies 173O80-67-6 SAC311 SYTO 13 488 SO6 LifeTechnologies 173O80-69-8 SAC312 SYTO 16 488 520 LifeTechnologies 173O80-72-3 SAC313 SYTO 17 618 637 LifeTechnologies 189233-66-7 SAC314 SYTO 45 450 486 LifeTechnologies 33SO78-86-9 SAC315 SYTO 59 622 643 LifeTechnologies 23S422-34-1 SAC316 SYTO 60 6SO 681 LifeTechnologies 33SO79-14-6 SAC317 SYTO 61 618 651 LifeTechnologies 335079-15-7 SAC318 SYTO 62 6SO 681 LifeTechnologies 286951-08-4 SAC319 SYTO 82 S4O S60 LifeTechnologies 33SO79-10-2 SAC32O SYTO 9 482 500 LifeTechnologies 208.540-89-O SAC321 SYTOXAADwanced S46 646 LifeTechnologies No names found SAC322 SYTOX Blue 431 480 LifeTechnologies 396O77-OO-2 SAC323 SYTOX Green SO4 523 LifeTechnologies 1941 OO-76-O SAC324 SYTOX Orange 547 570 LifeTechnologies 324767-53-5 US 9,714,897 B2 29 30 TABLE 2-continued

Exci- Emis D NAME Alternate Names tation sion Vendorf Source ACS CASii SAC325 SYTOX Red 640 658 LifeTechnologies 915152-67-9 SAC326 to Tomato 554 581 Contech 1114838-94-6 SAC334 Tetramethylrhodamine TMRho 553 581 LifeTechnologies 70281-37-7 SAC329 Texas Red Texas Red (R)-X, succinimidyl ester; 589 615 LifeTechnologies 82354-19-6 C41H44N4O1 OS2 SAC330 Texas Red-X, succinimidyl 589 615 LifeTechnologies 21 6972-99-5 ester SAC331 Thiazole Orange 500 530 Sigma Aldrich 107091-89-4 SAC332 ThiolTracker Violet 4O6 526 LifeTechnologies No names found SAC335 TO-PRO-1 TO-PRO (R)-1 iodide (515/531); 509 533 LifeTechnologies 1571.99-59-2 methyl-2(3H)- benzothiazolylidene)methyl-1-3- (trimethylammonio)propyl-, diiodide?: SAC338 TO-PRO-3 TO-PRO (R)-3 iodide (642/661); 642 661 LifeTechnologies 1571.99-63-8 methyl-2(3H)-benzothiazolylidene)-1- propenyl)-1-3- (trimethylammonio)propyl-, diiodide? ISAC341 TOTO-1 TOTO (R)-1 iodide (514/533): 509 533 LifeTechnologies 143413-84-7 propanediylbis(dimethyliminio)-3,1- propanediyl)bis(4-(3-methyl-2(3H)- benzothiazolylidene)methyl-, tetraiodide? ISAC344 TOTO-3 TOTO (R)-3 iodide (642/660); 642 661 LifeTechnologies 166196-17-4 CS3H624N6S2 ISAC346 Tricolor 563 670 LifeTechnologies 478.184-SO-8 ISAC347 TRITC Tetramethylrhodamine; 547 572 LifeTechnologies 745735-42-6 tetramethylrhodamine-5-(and-6)- Xanthylium, 9-(2- carboxyisothiocyanatophenyl)-3,6- bis(dimethylamino)-, inner salt ISAC3S1 TruRed 490 695 Not Commercialized 396O76-95-2 ISAC352 V19 397 572 Not Commercialized No names found ISAC3S3 V450 40S 448 BDBioscences 1257844-82-8 ISAC3S4 V500 415 500 BDBioscences 13331 60-12-5 ISAC3SS VioBlue 400 452 Miltenyl Biotech 1431147-59-9 ISAC356 VioGreen 388 520 Miltenyl Biotech No names found ISAC357 Vybrant DyeCycle Green 505 535 LifeTechnologies 1431152-SO-9 ISAC358 Vybrant DyeCycle Orange 518 563 LifeTechnologies 105.5990-89-0 ISAC359 Vybrant DyeCycle Ruby 637 686 LifeTechnologies 13452O2-72-3 ISAC360 Vybrant DyeCycle Violet 370 436 LifeTechnologies 101S439-88-9 ISAC361 YFP Yellow Fluorescent Protein 505 530 Contech No names found ISAC363 YO-PRO-1 YO-PRO (R)-1 iodide (491/509); 491 506 LifeTechnologies 1S2O68-09-2 C24H29I2N3O ISAC36S YO-PRO-3 YO-PRO (R)-3 iodide (612/631): 613 629 LifeTechnologies 1571.99-62-7 methyl-2(3H)-benzoxazolylidene)-1- propenyl)-1-3- (trimethylammonio)propyl-, diiodide? ISAC368 YOYO-1 YOYO (R)-1 iodide (491/509); 491 509 LifeTechnologies 143413-85-8 C49H58I4N6O2: ISAC370 YOYO-3 YOYO (R)-3 iodide (612/631): 613 629 LifeTechnologies 156312-20-8 propanediylbis(dimethyliminio)-3,1- propanediyl)bis(4-3-(3-methyl-2(3H)- benzoxazolylidene)-1-propenyl-, tetraiodide?: ISAC373 ZsCreen 494 517 Contech 1216871-88-3

55 Commercially available beads including, but not limited um)), red lasers (catalog no. A-16501 (2.5 um), A-16504 to, those sold by Bangs Laboratories, Inc, Sperhotech Inc., (6.0 um)) or UV lasers (catalog no. A-16502 (2.5 um), Thermo Scientific, Inc. and equivalent Suppliers) can be A-16505 (6.0 m)) can be embedded in on or more of the used in combination with the hydrogel particles described hydrogel particles provided herein. herein. Depending on the assay, it is within the ordinary skill 60 In one embodiment, a fluorescent bead that can be excited in the art to select a bead with the proper bead diameter, at any wavelength from 365 nm.-650 nm is embedded in a fluorescent emission and/or excitation spectrum and/or fluo hydrogel particle. In one embodiment, the bead is a “rain rescent intensity. For example, a quality control bead used in bow particle' that contains a mixture of fluorophores, for conjunction with a blue, red or UV laser can be embedded example 4 fluorophores, 5 fluorophores, 6 fluorophores, into one or more hydrogel particles provided herein. For 65 seven fluorophores or eight fluorophores. In this regard, the example, an AlignflowTM flow cytometry alignment bead for user selects which wavelength to excite the particle, depend blue lasers (catalog no. A-16500 (2.5 m), A-16503 (6.0 ing on the fluorophore being interrogated. Rainbow particles US 9,714,897 B2 31 32 are commercially available, for example, from BD Biosci cell, fibroblast or mesenchymal cell. Hydrogel particles in ences (catalog nos. 556298 (mid range FL1 fluorescence), one embodiment, are tuned to have one or more optical 556286 (6 color, 3.0-3.4 um), 55.6288 (6 color, 6.0–6.4 um), properties Substantially similar to a professional phagocyte 559123 (8 color)) and Spherotech in various diameters (e.g., set forth in Table 3 below (prior to and/or after particle catalog nos. RCP20-5 (4 color), RCP-30-5 (6 peaks), RCP uptake). 30-5A (8 peaks) A cell sorting set-up bead can be embedded in one or more TABLE 3 of the hydrogel particles provided herein. In one embodi ment, a cell sorting set-up beads approximates the size, Location Phagocyte type emission wavelength, and intensity of a biological sample, 10 Blood Neutrophil, monocyte and can be used to calibrate a flow cytometer's cell sorting Bone marrow Macrophage, monocyte, sinusoidal cell, lining cell system, including laser source, optics, and stream flow. In Bone tissue Osteoclast Gut and intestinal Macrophage one embodiment, a cell sorting set-up beads is embedded in Peyer's patches one or more hydrogel particles and is amenable for use with Connective tissue Histiocyte, macrophage, monocyte, dendritic cell a UV, blue, green/yellow or red laser. Where a green laser is 15 Liver Kupffer cell, monocyte used, in one embodiment, the embedded bead is excited at Lung Self-replicating macrophage, monocyte, mast cell, dendritic cell 570 nm with emission of 575 nm, but may also be exited at Lymphoid tissue Free and fixed macrophages and monocytes, dendritic 488 nm. Commercially available cell sorting set-up beads cell are available, for example, from Life Technologies (catalog Nervous tissue Microglial cell (CD4+) nos. C-16506 (UV laser), C-16508 (blue laser), C-16509 Spleen Free and fixed macrophages, monocytes, sinusoidal cell (green-yellow laser), C-16507 (red laser)). Thymus Free and fixed macrophages, monocytes A compensation control bead can also be embedded in Skin Resident Langerhans cells, dendritic cells, one or more of the hydrogel particles provided herein. conventional macrophage, mast cell Accurate compensation is an important parameter for effec tive multicolor analysis in flow cytometry. However, cellu 25 lar-based compensation controls are not completely effective In one embodiment, a plurality of hydrogel particles of the as many antigens are not highly expressed, and dimly invention, embedded with a Substance Such as nucleic acid stained cells can lead to inaccurate compensation settings. or a bead is used as control reagents for a genomic cytometry A compensation control bead, in one embodiment, assay. In this regard, a specific number of copies of a includes a fluorescent antibody conjugate capture capacity 30 particular chromosome, RNA sequence and/or DNA (positive compensation bead) or is inert (negative compen sequence can be mimicked by the embedded substance. The sation bead). The compensation bead is mixed with a hydrogel particle can then be used as a control for a sample fluorophore-conjugated human, mouse, rat, hamster, or rab being probed for genetic information, Such as the number of bit antibody; the two components provide a distinct high copies of a chromosome, the number of copies of an RNA signal positive control with an appropriate negative popu 35 sequence and/or the number of copies of an RNA sequence. lation that can then be used to set compensation properly The three primary modes of deconvolution for flow regardless of the intensity of the cells in the actual experi cytometry are the two passive optical properties of a particle ment. Once the antibody is mixed with the bead, it is (forward scattering, FSC, corresponding to the refractive embedded in one or more of the hydrogel particles provided index, or RI; and side scattering, SSC) and biomarkers herein. Commercially available compensation beads are 40 present on the Surface of a given cell type. Therefore, available, for example, from Life Technologies (catalog nos. compositions that allow hydrogel particles of the disclosure A-10344, A-10389, A10497, A10513) and Spherotech (cata to mimic specific cell types with respect to these three modes log nos. CMIg-P-08-2K, CMIg-P-30-2K, CMIg-P-50-3K, are useful for providing synthetic, robust calibrants for flow CMIg-P-70-3K). cytometry. In one embodiment, a hydrogel particle with an embed 45 In one embodiment, the refractive index (RI) of a dis ded/encapsulated bead is used as a reference for a cellular closed hydrogel particle is greater than about 1.10, greater assay, for example, a phagocytosis assay cytoxicity assay, than about 1.15, greater than about 1.20, greater than about motility assay, viability assay, etc. Phagocytosis is the pro 1.25, greater than about 1.30, greater than about 1.35, cess by which a cell engulfs a solid particle to form an greater than about 1.40, greater than about 1.45, greater than internal vesicle known as a phagosome. In this regard, a 50 about 1.50, greater than about 1.55, greater than about 1.60, hydrogel particle can be tuned to have one or more optical greater than about 1.65, greater than about 1.70, greater than properties Substantially similar to a phagocyte, before and about 1.75, greater than about 1.80, greater than about 1.85, after the phagocyte engulfs a particle. Accordingly, in one greater than about 1.90, greater than about 1.95, greater than embodiment, the hydrogel particles provided herein are used about 2.00, greater than about 2.10, greater than about 2.20, as control particles for a phagocytosis assay. In a further 55 greater than about 2.30, greater than about 2.40, greater than embodiment, (i) one or more of the optical properties of a about 2.50, greater than about 2.60, greater than about 2.70. hydrogel particle is Substantially similar to a phagocyte prior greater than about 2.80, or greater than about 2.90. to particle uptake and (ii) one or more of the optical In another embodiment, the refractive index (RI) of a properties of a second hydrogel particle is substantially disclosed hydrogel particle is about 1.10 to about 3.0, or similar to a phagocyte after to particle uptake. In this regard, 60 about 1.15 to about 3.0, or about 1.20 to about 3.0, or about a control is generated for measuring particle uptake by a 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 phagocyte. to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about In one embodiment, the phagocyte is a professional 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or phagocyte. In another embodiment, the phagocyte is a about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 non-professional phagocyte (i.e., a cell that consumes dying 65 to about 3.0, or about 2.0 to about 3.0. cells and foreign organisms). In a further embodiment, the In some embodiments, the refractive index (RI) of a non-professional phagocyte is an epithelial cell, endothelial disclosed hydrogel particle is less than about 1.10, less than US 9,714,897 B2 33 34 about 1.15, less than about 1.20, less than about 1.25, less to be bound by theory, the ability to selectively tune both than about 1.30, less than about 1.35, less than about 1.4.0, forward and side scatter of a hydrogel, as described herein, less than about 1.45, less than about 1.50, less than about allows for a robust platform to mimic a vast array of cell 1.55, less than about 1.60, less than about 1.65, less than types. about 1.70, less than about 1.75, less than about 1.80, less Although the invention is mainly described with respect than about 1.85, less than about 1.90, less than about 1.95, to the modification of optical properties, the invention is not less than about 2.00, less than about 2.10, less than about limited thereto. For example, hydrogel particles can be 2.20, less than about 2.30, less than about 2.40, less than fabricated and adjusted to tune the capacitance of the about 2.50, less than about 2.60, less than about 2.70, less particles, e.g., to calibrate coulter counters. In one embodi 10 ment, a hydrogel particle's capacitance is adjusted by alter than about 2.80, or less than about 2.90. ing the amount of hydrogel monomer in the composition. The SSC of a disclosed hydrogel particle is most mean For example, polyanaline, polyacetylene; polyphenylene ingfully measured in comparison to that of target cell. In vinylene; polypyrrole (X—NH) and polythiophene (X=S) Some embodiments, a disclosed hydrogel particle has an co-monomers; and polyaniline (X—NH/N) and polyphe SSC within 30%, within 25%, within 20%, within 15%, 15 nylene Sulfide (X=S) co-monomer concentrations can all be within 10%, within 5%, or within 1% that of a target cell, as adjusted to alter capacitance. In one embodiment, the con measured by a cytometric device. centration of one or more of these monomers is increased to The SSC of a hydrogel particle in one embodiment, is increase the capacitance of the hydrogel particle. modulated by incorporating a high-refractive index mol In some embodiments, a hydrogel particle of the disclo ecule (or plurality thereof) in the hydrogel. In one embodi Sure has material modulus properties (e.g., elasticity) more ment, a high-refractive index molecule is provided in a closely resembling that of a target cell as compared to a hydrogel particle, and in a further embodiment, the high polystyrene bead of the same diameter. refractive index molecule is colloidal silica, alkyl acrylate, After the hydrogel particle is formed, one or more of the alkyl methacrylate or a combination thereof. Thus in some particle's Surfaces can be functionalized, for example, to embodiments, a hydrogel particle of the disclosure com mimic one or more optical properties of a target cell or a prises alkyl acrylate and/or alkyl methacrylate. Concentra 25 labeled target cell. The functionalized hydrogel particle can tion of monomer in one embodiment is adjusted to further also include an embedded bead or Substance Such as a adjust the refractive index of the hydrogel particle. biomolecule, as described above. In one embodiment, one or Alkyl acrylates or Alkyl methacrylates can contain 1 to more hydrogel particles are functionalized with one or more 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group, Such fluorescent dyes, one or more cell Surface markers (or as methyl, ethyl, n-propyl, isopropyl. n-butyl, isobutyl or 30 epitope binding regions thereof), or a combination thereof. tertbutyl, 2-ethylhexyl, heptyl or octyl groups. The alkyl In one embodiment, the hydrogel particle is formed by group may be branched or linear. polymerizing at least one bifunctional monomer and after High-refractive index molecules can also include vinylar formation, the hydrogel particle includes one or more func enes such as styrene and methylstyrene, optionally Substi tional groups that can be used for further attachment of a cell tuted on the aromatic ring with an alkyl group. Such as 35 Surface marker, an epitope binding region of a cell Surface methyl, ethyl or tert-butyl, or with a halogen, Such as marker, a fluorescent dye, or combination thereof. The free chlorostyrene. functional group, in one embodiment, is an amine group, a In some embodiments, FSC is modulated by adjusting the carboxyl group, a hydroxyl group or a combination thereof. percentage of monomer present in the composition thereby Depending on the functionalization desired, it is to be altering the water content present during hydrogel forma understood that multiple bifunctional monomers can be tion. In one embodiment, where a monomer and co-mono 40 used, for example, to functionalize the particle using differ mer are employed, the ratio of monomer and co-monomer is ent chemistries and with different molecules. adjusted to change the hydrogel particle's forward Scatter A hydrogel particle can be functionalized with any fluo properties. This is shown in both FIG. 11 and FIG. 12. rescent dye known in the art, including fluorescent dyes The FSC of a disclosed hydrogel particle is most mean listed in The MolecularProbes(R Handbook-A Guide to ingfully measured in comparison to that of target cell. In 45 Fluorescent Probes and Labeling Technologies, incorporated Some embodiments, a disclosed hydrogel particle has an herein by reference in its entirety for all purposes. Func FSC within 30%, within 25%, within 20%, within 15%, tionalization can be mediated by a compound comprising a within 10%, within 5%, or within 1% that of a target cell, as free amine group, e.g. allylamine, which can be incorporated measured by a cytometric device. into a bifunctional monomer used to form the hydrogel, as FSC is related to particle volume, and thus can be 50 discussed above. modulated by altering particle diameter, as described herein. Non-limiting examples of known fluorescent dyes that Generally, it has been observed that large objects refract can be used to functionalize the Surface of a hydrogel more light than Smaller objects leading to high forward particle described herein include: 6-carboxy-4',5'-dichloro scatter signals (and vice versa). Accordingly, particle diam 2,7-dimethoxyfluorescein succinimidylester, 5-(and-6)-car eter in one embodiment is altered to modulate FSC proper boxyeosin; 5-carboxyfluorescein; 6 carboxyfluorescein; ties of a hydrogel particle. For example, hydrogel particle 55 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5- diameter is increased in one embodiment is altered by carboxymethoxy-2-nitrobenzyl)ether.-alanine-carboxamide, harnessing larger microfluidic channels during particle for or Succinimidyl ester, 5-carboxyfluoresceinsuccinimidyl mation. ester; 6-carboxyfluorescein succinimidyl ester, 5-(and-6)- SSC can be engineered by encapsulating nanoparticles carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotri within hydrogels to mimic organelles in a target cell. In 60 azinyl)amino fluorescein; 2, 7-difluorofluorescein; eosin Some embodiments, a hydrogel particle of the disclosure 5-isothiocyanate; erythrosin5-isothiocyanate; comprises one or more types of nanoparticles selected from 6-(fluorescein-5-carboxamido) hexanoic acid or Succinim the group consisting of polymethyl methacrylate (PMMA) idyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic nanoparticles, polystyrene (PS) nanoparticles, and silica acid or succinimidylester; fluorescein-S-EX succinimidyl nanoparticles. See also FIGS. 11 and 12 which show that 65 ester; fluorescein-5-isothiocyanate; fluorescein-6-isothio addition of various concentrations of nanoparticles allow for cyanate; OregonGreen R. 488 carboxylic acid, or succinim the adjustment of side scatter of a particle. Without wishing idyl ester; Oregon Green(R) 488 isothiocyanate; Oregon US 9,714,897 B2 35 36 Green R. 488-X succinimidyl ester; Oregon Green(R) 500 nyl-1,3butadienyl)-4-bora-3a,4a-diaza-S-indacene-3-propi carboxylic acid; Oregon Green R. 500 carboxylic acid, suc onicacid Succinimidyl ester, 4.4-difluoro-5-(2-pyrrolyl)-4- cinimidylester or triethylammonium salt; Oregon Green(R) bora-3a,4a-diaza-S-indacene-3-propionic acid Succinimidyl 514 carboxylic acid; Oregon Green R 514 carboxylic acid or ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s- succinimidyl ester; RhodamineGreenTM carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine GreenTM indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or Suc carboxylic acid, trifluoroacetamide or Succinimidylester, cinimidyl ester, 4.4-difluoro-5-styryl-4-bora-3a, 4a-diaza-S- Rhodamine GreenTM-X succinimidyl ester or hydrochloride: indacene-3-propionic acid; 4.4-difluoro-5-styryl-4-bora-3a, RhodolgreenTM carboxylic acid, N.O-bis-(trifluoroacetyl) or 4a-diaza-Sindacene-3-propionic acid; Succinimidyl ester; Succinimidylester, bis-(4-carboxypiperidinyl) sulfonerhod 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4adiaza-s-in amine or di(Succinimidylester), 5-(and-6)carboxynaphtho 10 dacene-8-propionicacid; 4.4-difluoro-1,3,5,7-tetramethyl fluorescein, 5-(and-6)carboxynaphthofluorescein Succinim 4bora-3a,4a-diaza-Sindacene-8-propionicacid Succinimidyl idyl ester; 5-carboxyrhodamine 6G hydrochloride: ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sin 6-carboxyrhodamine6Ghydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinim dacene-3-propionicacid Succinimidyl ester, 6-(((4-(4.4-dif idyl ester; 5-(and-6)-carboxyrhodamine(5G succinimidyl luoro-5-(2-thienyl)-4-bora-3a,4adiaZaS-indacene-3-yl)phe ester, 5-carboxy-2',4',5'7"-tetrabromosulfonefluorescein 15 noxy)acetyl)amino)hexanoic acid or Succinimidyl ester; and Succinimidyl esteror bis-(diisopropylethylammonium) salt; 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-in 5-carboxytetramethylrhodamine; 6-carboxytetramethylrho dacene-3-yl) styryloxy)acetyl)aminohexanoic acid or Suc damine; 5-(and-6)-carboxytetramethylrhodamine; 5-car cinimidyl ester. boxytetramethylrhodamine succinimidyl ester; 6-carboxyte tramethylrhodaminesuccinimidyl ester, 5-(and -6)- Fluorescent dyes for derivatization of the surface of one carboxytetramethylrhodamine Succinimidyl ester, or more hydrogel particles in one embodiment, include, but 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine Succin are not limited to, Alexafluor dyes commercially available imidyl ester; 6-carboxy-Xrhodamine succinimidyl ester; from Invitrogen, including but not limited to Alexa Fluor R 5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester; 5-car 350 carboxylic acid; Alexa Fluorr) 430 carboxylic acid; boxy-X-rhodamine triethylammonium salt; LissamineTM Alexa Fluor R. 488 carboxylic acid; Alexa Fluor R. 532 car rhodamine B Sulfonyl chloride; malachite green; isothiocya 25 boxylic acid; Alexa Fluorr 546 carboxylic acid; Alexa nate; NANOGOLDR) mono(sulfosuccinimidyl ester); Fluor R. 555 carboxylic acid; Alexa Fluor R. 568 carboxylic QSYR 21 carboxylic acid or succinimidyl ester; QSYR 7 acid; Alexa Fluor R 594 carboxylic acid; Alexa Fluor R 633 carboxylic acid or succinimidyl ester; Rhodamine RedTM-X carboxylic acid; Alexa Fluor R 647 carboxylic acid; Alexa Succinimidyl ester, 6-(tetramethylrhodamine-5-(and-6)-car Fluorr 660 carboxylic acid; and Alexa Fluor R 680 carbox boxamido) hexanoic acid; Succinimidyl ester, tetramethyl 30 ylic acid. In another embodiment, fluorescent dyes for use rhodamine-5-isothiocyanate; tetramethylrhodamine-6-isoth with the hydrogel particles and methods described herein iocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; include cyanine dyes commercially available from Amer Texas Red R) sulfonyl: Texas Red R) sulfonyl chloride; Texas sham-Pharmacia Biotech, including, but not limited to Cy3 Red R-X STP ester or sodium salt, Texas Red R-X succin NHS ester; Cy5 NHS ester; Cy5.5 NHSester; and Cy7 NHS imidyl ester, Texas Red R-X succinimidyl ester; and X-rho 35 ester. damine-5-(and-6) isothiocyanate. Other examples of fluorescent dyes for use with the It is within the ordinary skill in the art to select a suitable hydrogel particles described herein include, but are not dye or dyes based on the desired spectral excitation and limited to, BODIPYR dyes commercially available from emission properties of the hydrogel particle. Invitrogen, including, but not limited to BODIPYR FL; 40 Hydrogel particles, in one embodiment, are functional BODIPYR TMR STP ester; BODIPYR TR-X STP ester; ized with one or more cell Surface markers (see, e.g., Tables BODIPYR 630/650-X STPester; BODIPYR 650/665-X 4 and 7-8), or fragments thereof, for example, extracellular STP ester; 6-dibromo-4, 4-difluoro-5,7-dimethyl-4-bora-3a, portions thereof in the case of transmembrane proteins, for 4a-diaza-S-indacene-3-propionic acid Succinimidyl ester, example, by attaching the one or more cell Surface markers, 4,4-difluoro-4-bora-3a.4a-diaza-S-indacene-3,5-dipropionic 45 extracellular portions or ligand binding regions thereof to acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-in the particle via a free amine, free carboxyl and/or free dacene-3-pentanoicacid; 4,4-difluoro-5,7-dimethyl-4- hydroxyl group present on the Surface of the hydrogel bora3a,4a-diaza-S-indacene-3-pentanoicacid Succinimidyl particle. Functionalization of a hydrogel particle with a dye ester; 4.4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-in or cell Surface molecule can also occur through a linker, for dacene-3propionicacid; 4.4-difluoro-5,7-dimethyl-4-bora 50 example a streptavidin/biotin conjugate. 3a,4adiaza-S-indacene-3-propionicacid Succinimidyl ester, Depending on the target cell, individual hydrogel particles 4.4difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene can be derivatized with one or more cell surface markers, or 3propionic acid; Sulfo Succinimidyl ester or Sodium salt; fragments thereof, for example, extracellular portions 6-((4.4-difluoro-5,7-dimethyl-4-bora-3a.4a-diaza-s-in thereof in the case of transmembrane proteins to further dacene-3propionyl)amino) hexanoic acid; 6-((4.4-difluoro 55 mimic the structural properties of the target cell. Tables 4 5, 7 dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionyl) and 7-8, provided below, sets forth a non-limiting list of cell amino) hexanoic acid or Succinimidyl ester, N-(4, 4-difluoro Surface markers that can be used to derivative hydrogel 5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionyl) particles, depending on the target cell. Although the cell cysteic acid, Succinimidyl ester or triethylammonium salt; Surface marker is provided, it is understood that a portion of 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4- 60 the cell Surface marker, for example, a receptor binding bora3a, 4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza portion, a ligand binding portion, or an extracellular portion sindacene-3-propionicacid; 4.4-difluoro-5, 7-diphenyl-4- of the marker can be used to derivative the hydrogel particle bora3a, 4a-diaza-S-indacene-3-propionic acid Succinimidyl (at the free functional group, as described above). See also ester, 4.4-difluoro-5-phenyl-4-bora-3a,4a-diaza-S-indacene FIGS. 11 and 12 which show that hydrogel surface modi 3-propionic acid; Succinimidyl ester, 6-((4.4-difluoro-5-phe 65 fication with for example, a cell Surface receptor, together nyl-4 bora-3a,4a-diaza-S-indacene-3-propionyl)amino) with the selective tuning of FSC and/or SSC, allows for the hexanoicacid or Succinimidyl ester, 4.4-difluoro-5-(4-phe fabrication of a hydrogel particle with the desired feature(s). US 9,714,897 B2 37 38 TABLE 4 Target Cell Cell Surface Marker(s) (human) Cell Surface Marker(s) (mouse) B Cell CD19, CD20 D19, CD22 (B cell activation arker), CD45R/B220 T Cell CD3, CD4, CD8 D3, CD4, CD8 Activated T Cells CD25, CD69 D25, CD69 Dendritic Cell CD1c, CD83, CD123, CD141, D11c, CD123, MHC II CD209, MHC II Plasmacytoid CD123, CD303, CD304 D11c, CD317 Dendritic Cells* Platelet (resting) CD42b D41 Platelet (activated) CD62P D62P Natural Killer Cells CD16, CD56 D49b (clone DX5) Hematopoietic Stem CD34, CD90 D48, CD117, CD150, Sca-1 Cell Macrophage CD11b, CD68, CD163 F4/80, CD68 Monocyte CD14, CD16, CD64 D11b, CD115, Ly-6C Plasma Cell CD138 D138 Red Blood Cell CD235a ER-119 Neutrophil CD15, CD16 D11b, Ly-6B.2, Ly6G, Gr-1 Basophil 2D7 antigen, CD123, CD203c, D20OR3, Fce RIC. FceRIO. Eosinophil CD11b, CD193, EMR1, Siglec-8 D11b, CD193, F4/80, Siglec-F Granulocyte CD66b D66b, Gr-1/Ly6G, Ly6C Endothelial cell CD146 D146 MECA-32, CD106, CD31, D62E (activated endothelial cell) Epithelial cell CD326 D326 (EPCAM1) Natural Killer (NK) CD56 D335 (NKp46) cell Myeloid derived CD11b, CD14, CD33 (Siglec-3) C D11b, GR1 Suppressor cell (MDSC)

30 Cell types including but not limited to various cell lines TABLE 5-continued such as CHO, HEK-293, BHK-21, NSO, MDCK, VERO, cell of Brunner's gland in duodenum MRC-S, W1-38 and Sp2/0 Mouse Myeloma (hybridomas). cell of seminal vesicle Table 5 and Table 6 each provides other cell types for use cell of prostate gland with the hydrogel particles described herein. 35 cell of bulbourethral gland cell of Bartholin's gland cell of gland of Littre TABLE 5 cell of endometrium of uterus isolated goblet cell of respiratory and digestive tracts keratinocyte of epidermis mucous cell of lining of stomach basal cell of epidermis zymogenic cell of gastric gland keratinocyte of fingernails and toenails 40 Oxyntic cell of gastric gland basal cell of nail bed acinar cell of pancreas hair shaft cells Paneth cell of small intestine medullary hair shaft cells type II pneumocyte of lung cortical hair shaft cells Clara cell of lung cuticular hair shaft cells cells of anterior pituitary hair-root sheath cells 45 cell of intermediate pituitary cuticular hair-root sheath cells cells of posterior pitutiary hair-root sheath cells of Huxley's layer cells of gut and respiratory tract hair-root sheath cells of Henle’s layer cells of thyroid gland external hair-root sheath cells cells of parathyroid gland hair matrix cell (stem cell) cells of adrenal gland Surface epithelial cell of stratified squamous epithelium of tongue 50 steroid hormones Surface epithelial cell of stratified squamous epithelium of oral cavity cells of gonads Surface epithelial cell of stratified squamous epithelium of esophagus cells of juxtaglomerular apparatus of kidney Surface epithelial cell of stratified squamous epithelium of anal canal juxtaglomerular cell surface epithelial cell of stratified squamous epithelium of distal urethra macula Surface epithelial cell of stratified squamous epithelium of vagina densa cell basal cell of these epithelia 55 peripolar cell cel of urinary epithelium cells of salivary gland mesangial cell Mucous cells of Salivary gland brush border cell of intestine Serous cell of Salivary gland striated duct cell of exocrine glands cell of von Ebner's gland in tongue gall bladder epithelial cell cell of mammary gland brush border cell of proximal tubule of kidney cell of lacrimal gland 60 distal tubule cell of kidney cell of ceruminous gland of ear nonciliated cell of ductulus efferens cell of eccrine Sweat gland epididymal principal cell cell of eccrine Sweat gland epididymal basal cell cell of apocrine Sweat gland hepatocyte cell of gland of Moll in eyelid white fat cell cel of sebaceous gland 65 brown fat cell cel of Bowman's gland in nose lipocyte of liver US 9,714,897 B2 39 40 TABLE 5-continued TABLE 5-continued type I pneumocyte Schwann cell pancreatic duct cell satellite cell (encapsulating peripheral nerve cell bodies) parietal cell of kidney glomerulus enteric glial cell podocyte of kidney glomerulus leOS cell of thin segment of loop of Henle glial cells collecting duct cell (in kidney) anterior lens epithelial cell duct cell of seminal vesicle lens fiber (crystallin-containing cell) duct cell of prostate gland melanocyte vascular endothelial cells of blood vessels and lymphatics retinal pigmented epithelial cell fenestrated vascular endothelial cells 10 Oogonium oocyte continuous vascular endothelial cells spermatocyte splenic vascular endothelial cells spermatogonium (stem cell for spermatocyte) synovial cell ovarian follicle cell Serosal cell Sertoli cell (in testis) Squamous cell lining perilymphatic space of ear thymus epithelial cell cells lining endolymphatic space of ear Salivary gland mucous cell Squamous cell 15 Salivary gland number 1 columnar cells of endolymphatic sac Von Ebner's gland cell in tongue “dark cell Mammary gland cell vestibular membrane cell Lacrimal gland cell stria vascularis basal cell Ceruminous gland cell in ear stria vascularis marginal cell Eccrine Sweat gland dark cell cell of Claudius Eccrine Sweat gland clear cell cell of Boettcher Apocrine Sweat gland cell choroid plexus cell Gland of Moll cell in eyelid Squamous cell of pia-arachnoid Sebaceous gland cell cells of ciliary epithelium of eye Bowman's gland cell in nose corneal “endothelial cell Brunner's gland cell in duodenum Ciliated Cells of respiratory tract 25 Seminal vesicle cell Ciliated Cells of oviduct and of endometrium of uterus Prostate gland cell Ciliated Cells of rete testis and ductulus efferens Bulbourethral gland cell Ciliated Cells of central nervous system Bartholin's gland cell epithelial Gland of Littre cell ameloblast Uterus endometrium cell nonepithelial 30 goblet cell of respiratory and digestive tracts chondrocytes Stomach lining mucous cell osteoblastfosteocyte Gastric gland zymogenic cell osteoprogenitor cell Gastric gland oxyntic cell hyalocyte of vitreous body of eye Pancreatic acinar cell stellate cell of perilymphatic space of ear Paneth cell of small intestine skeletal muscle cells 35 pneumocyte of lung heart muscle cells Clara cell of lung Smooth muscle cells (various) anterior pituitary cells myoepithelial cells Somatotropes red blood cell Lactotropes megakaryocyte Thyrotropes macrophages and related cells Gonadotropes neutrophil 40 Corticotropes eosinophil melanocyte-stimulating hormone basophil Magnocellular neurosecretory cells Secreting: mast cell Gut and respiratory tract cells secreteing: T lymphocyte Thyroid gland cells B lymphocyte thyroid epithelial cell photoreceptors (rods, cones, and can be blue sensitive, green sensitive, 45 parafollicular cell red sensitive) Parathyroid gland cells inner hair cell of organ of Corti Parathyroid chief cell outer hair cell of organ of Corti Oxyphil cell type I hair cell of vestibular apparatus of ear Adrenal gland cells type II hair cell of vestibular apparatus of ear chromaffin cells type II taste bud cell 50 Secreting steroid hormones (mineralcorticoids and gluco corticoids) olfactory neuron Leydig cell of testes Secreting testosterone basal cell of olfactory epithelium Theca interna cell of ovarian follicle Secreting estrogen carotid body cell type I Corpus luteum cell of ruptured ovarian follicle secreting progesterone carotid body cell type II Granulosa lutein cells Merkel cell of epidermis Theca lutein cells primary sensory neurons specialized for touch (various) 55 Juxtaglomerular cell (renin secretion) primary sensory neurons specialized for temperature - cold sensitive Macula densa cell of kidney primary sensory neurons specialized for temperature - heat sensitive Peripolar cell of kidney primary sensory neurons specialized for pain (various) Mesangial cell of kidney proprioceptive primary sensory neurons (various) epidermal keratinocyte Autonomic Neurons Epidermal basal cell inner pillar cell Keratinocyte of fingernails and toenails outer pillar cell 60 Nail bed basal cell (stem cell) inner phalangeal cell Medullary hair shaft cell outer phalangeal cell Cortical hair shaft cell border cell Cuticular hair shaft cell Hensen cell Cuticular hair root sheath cell Supporting cell of vestibular apparatus Hair root sheath cell of Huxley's layer Supporting cell of taste bud (type I taste bud cell) 65 Hair root sheath cell of Henle’s layer Supporting cell of olfactory epithelium External hair root sheath cell US 9,714,897 B2 41 42 TABLE 5-continued TABLE 5-continued Hair matrix cell (stem cell) Loose connective tissue fibroblasts epithelial cell of stratified squamous epithelium of cornea, Corneal fibroblasts (corneal keratocytes) epithelial cell of stratified squamous epithelium of tongue Tendon fibroblasts epithelial cell of stratified squamous epithelium of oral cavity 5 Bone marrow reticular tissue fibroblasts epithelial cell of stratified squamous epithelium of esophagus nonepithelial fibroblasts epithelial cell of stratified squamous epithelium of anal canal Pericyte epithelial cell of stratified squamous epithelium of distallurethra Nucleus pulposus cell of intervertebral disc epithelial cell of stratified squamous epithelium of vagina Cementoblasticementocyte basal cell (stem cell) of epithelia of cornea Odontoblastodontocyte basal cell (stem cell) of epithelia of tongue 10 Hyaline cartilage chondrocyte basal cell (stem cell) of epithelia of oral cavity Fibrocartilage chondrocyte basal cell (stem cell) of epithelia of esophagus Elastic cartilage chondrocyte basal cell (stem cell) of epithelia of anal canal Osteoblastosteocyte basal cell (stem cell) of epithelia of distal urethra Osteoprogenitor cell basal cell (stem cell) of epithelia of vagina Hyalocyte of vitreous body of eye Urinary epithelium cell 15 Stellate cell of perilymphatic space of ear Auditory inner hair cell of organ of Corti Hepatic stellate cell (Ito cell) Auditory outer hair cell of organ of Corti Pancreatic stelle cell basal cell of olfactory epithelium skeletal muscle Cell Cold-sensitive primary sensory neurons Red skeletal muscle cell (slow) Heat-sensitive primary sensory neurons White skeletal muscle cell (fast) Merkel cell of epidermis (touch sensor) intermediate skeletal muscle cell Olfactory receptor neuron 20 nuclear bag cell of muscle spindle Pain-sensitive primary sensory neurons (various types) nuclear chain cell of muscle spindle Photoreceptor cells of retina in eye: Satellite cell (stem cell) Photoreceptor rod cells Heart muscle cells Photoreceptor blue-sensitive cone cell of eye Ordinary heart muscle cell Photoreceptor green-sensitive cone cell of eye Nodal heart muscle cell Photoreceptor red-sensitive cone cell of eye 25 Purkinje fiber cell Proprioceptive primary sensory neurons Smooth muscle cell Touch-sensitive primary sensory neurons Myoepithelial cell of iris Type I carotid body cell Myoepithelial cell of exocrine glands Type II carotid body cell Erythrocyte Type I hair cell of vestibular system of ear Megakaryocyte Type II hair cell of vestibular system of ear 30 Monocyte Type I taste bud cell Connective tissue macrophage Cholinergic neural cell Epidermal Langerhans cell Adrenergic neural cell Osteoclast (in bone) Peptidergic neural cell Dendritic cell (in lymphoid tissues) Inner pillar cell of organ of Corti Microglial cell (in central nervous system) Outer pillar cell of organ of Corti 35 Neutrophil granulocyte Inner phalangeal cell of organ of Corti Eosinophil granulocyte Outer phalangeal cell of organ of Corti Basophil granulocyte Border cell of organ of Corti Hybridoma cell Hensen cell of organ of Corti Mast cell Vestibular apparatus Supporting cell Helper T cell Taste bud Supporting cell Suppressor T cell Olfactory epithelium supporting cell 40 Cytotoxic T cell Schwann cell Natural Killer T cell Satellite glial cell B cell Enteric glial cell Natural killer cell Astrocyte Reticulocyte Neuron cells Stem cells and committed progenitors for the blood and immune Oligodendrocyte 45 system (various types) Spindle neuron Oogonium Oocyte Anterior lens epithelial cell Spermatid Crystallin-containing lens fiber cell S permatocyte Hepatocyte Spermatogonium cell Adipocytes (white fat cell, brown fat cell, liver lipocyte) p 9. Kidney parietal cell 50 Spematozoon Kidney glomerulus podocyte Ovarian follicle cell Kidney proximal tubule brush border cell Sertoli cell (in testis) Loop of Henle thin segment cell Thymus epithelial cell Kidney distal tubule cell Interstitial kidney cells Kidney collecting duct cell Type I pneumocyte 55 Pancreatic duct cell Nonstriated duct cell TABLE 6 principal cell intercalated cell Keratinizing Epithelial Cells Duct cell intestinal brush border cell keratinocyte of epidermis (=differentiating epidermal cell) Exocrine gland striated duct cell 60 basal cell of epidermis (stem cell) Gall bladder epithelial cell keratinocyte of fingernails and toenails Ductulus efferens nonciliated cell basal cell of nail bed (stem cell) Epididymal principal cell hair shaft cells Epididymal basal cell medullary Ameloblast epithelial cell cortical Planum semilunatum epithelial cell of vestibular system of ear 65 cuticular Organ of Corti interdental epithelial cell hair-root sheath cells US 9,714,897 B2 43 44 TABLE 6-continued TABLE 6-continued Cuticular root sheath cells macula densa cell (uncertain but probably related in root sheath cells of Huxley's layer peripolar cell { function; possibly involved in secretion root sheath cells of Henle’s layer mesangial cell of erythropoietin) external root sheath cells 5 Epithelial Absorptive Cells in Gut, Exocrine Glands, and Urogenital Tract hair matrix cell (stem cell) Cells of Wet Stratified Barrier Epithelia brush border cell of intestine (with microvilli) striated duct cell of exocrine glands Surface epithelial cell of stratified squamous epithelium of cornea, gall bladder epithelial cell tongue, oral brush border cell of proximal tubule of kidney cavity, esophagus, anal canal, distal urethra, vagina 10 distal tubule cell of kidney basal cell of these epithelia (stem cell) nonciliated cell of ductulus efferens cell of urinary epithelium (lining bladder and urinary ducts) epididymal principal cell Epithelial Cells Specialized for Exocrine Secretion epididymal basal cell Cells Specialized for Metabolism and Storage cells of salivary gland mucous cell (secretion rich in polysaccharide) 15 hepatocyte (liver cell) Serous cell (secretion rich in glycoprotein enzymes) fat cells cell of von Ebner's gland in tongue (secretion to wash over taste white fat buds) brown fat cell of mammary gland, Secreting milk lipocyte of liver cell of lacrimal gland, secreting tears Epithelial Cells Serving Primarily a Barrier Function, Lining cell of ceruminous gland of ear, Secreting wax the Lung, Gut, Exocrine Glands, and Urogenital Tract cell of eccrine Sweat gland, Secreting glycoproteins (dark cell) cell of eccrine Sweat gland, Secreting Small molecules (clear cell) type I pneumocyte (lining air space of lung) cell of apocrine Sweat gland (odoriferous secretion, sex-hormone pancreatic duct cell (centroacinar cell) sensitive) nonstriated duct cell of Sweat gland, Salivary gland, mammary cell of gland of Moll in eyelid (specialized Sweat gland) gland, etc. cell of Sebaceous gland, Secreting lipid-rich sebum (various) cell of Bowman's gland in nose (secretion to wash over olfactory 25 parietal cell of kidney glomerulus epithelium) podocyte of kidney glomerulus cell of Brunner's glan in duodenum, Secreting alkaline solution of cell of thin segment of loop of Henle (in kidney) mucus and enzymes collecting duct cell (in kidney) cell of Seminal vesicle, Secreting components of Seminal fluid, duct cell of seminal vesicle, prostate gland, etc. (various) including fructose (as fuel for Swimming sperm) Epithelial Cells Lining Closed Internal Body Cavities cell of prostate gland, secreting other components of seminal fluid 30 cell of bulbourethral gland, secreting mucus vascular endothelial cells of blood vessels and lymphatics cell of Bartholin's gland, secreting vaginal lubricant fenestrated cell of gland of Littre, secreting mucus continuous cell of endometrium of uterus, Secreting mainly carbohydrates splenic isolated goblet cell of respiratory and digestive tracts, Secreting synovial cell (lining joint cavities, secreting largely hyaluronic CS 35 acid) mucous cell of lining of stomach Serosal cell (lining peritoneal, pleural, and pericardial cavities) zymogenic cell of gastric gland, secreting pepsinogen Squamous cell lining perilymphatic space of ear oxyntic cell of gastric gland, Secreting HCl cells lining endolymphatic space of ear acinar cell of pancreas, secreting digestive enzymes and squamous cell bicarbonate columnar cells of endolymphatic sac Paneth cell of Small intestine, Secreting lysozyme with microvilli type II pneumocyte of lung, Secreting Surfactant 40 without microvilli Clara cell of lung (function unknown) “dark cell vestibular membrane cell Cells Specialized for Secretion of Hormones stria vascularis basal cell stria vascularis marginal cell cells of anterior pituitary, Secreting growth hormone, follicle cell of Claudius stimulating hormone, luteinizing hormone, prolactin, 45 cell of Boettcher adrenocorticotropic hormone, and/or thyroid-stimulating hormone choroid plexus cell (secreting cerebrospinal fluid) cell of intermediate pituitary, secreting melanocyte-stimulating Squamous cell of pia-arachnoid hormone cells of ciliary epithelium of eye cells of posterior pitutiary, Secreting oxytocin and/or vasopressin pigmented cells of gut and respiratory tract, Secreting serotonin, endorphin, nonpigmented Somatostatin, gastrin, secretin, cholecystokinin, insulin, glucagon, 50 corneal “endothelial cell and or bombesin Ciliated Cells with Propulsive Function cells of thyroid gland, secreting thyroid hormone Ciliated Cells of respiratory tract calcitonin Ciliated Cells of Oviduct and of endometrium of uterus (in female) cells of parathyroid gland, secreting Ciliated Cells of rete testis and ductulus efferens (in male) parathyroid hormone 55 Ciliated Cells of central nervous system (ependymal cell lining oxyphil cell (function unknown) brain cavities) cells of adrenal gland, Secreting Cells Specialized for Secretion of Extracellular Matrix epinephrine epithelial norepinephrine ameloblast (Secreting enamel of tooth) steroid hormones planum semilunatum cell of vestibular apparatus of ear mineralocorticoids 60 (secreting proteoglycan) glucocorticoids interdental cell of organ of Corti (Secreting tectorial cells of gonads, secreting “membrane' covering testosterone (Leydig cell of testis) hair cells of organ of Corti) estrogen (theca interna cell of ovarian follicle) nonepithelial (connective tissue) progesterone (corpus luteum cell of ruptured ovarian follicle) fibroblasts (various-of loose connective tissue, of cornea, of cells of juxtaglomerular apparatus of kidney 65 tendon, of reticular tissue of bone marrow, juxtaglomerular cell (secreting renin) etc.) US 9,714,897 B2 45 46 TABLE 6-continued TABLE 6-continued pericyte of blood capillary touch nucleus pulposus cell of intervertebral disc Merkel cell of epidermis cementoblasticementocyte (secreting bonelike cementum of primary sensory neurons specialized for touch (various) root of tooth) temperature odontoblastodontocyte (secreting dentin of tooth) primary sensory neurons specialized for temperature chondrocytes cold sensitive of hyaline cartilage heat sensitive of fibrocartilage pain of elastic cartilage primary sensory neurons specialized for pain (various) Osteoblastosteocyte 10 configurations and forces in musculoskeletal system Osteoprogenitor cell (stem cell of Osteoblasts) proprioceptive primary sensory neurons (various) hyalocyte of vitreous body of eye Autonomic Neurons stellate cell of perilymphatic space of ear Contractile Cells cholinergic (various) adrenergic (various) skeletal muscle cells 15 peptidergic (various) red (slow) Supporting Cells of Sense Organs and of Peripheral Neurons white (fast) intermediate Supporting cells of organ of Corti muscle spindle-nuclear bag inner pillar cell muscle spindle-nuclear chain outer pillar cell satellite cell (stem cell) inner phalangeal cell heart muscle cells outer phalangeal cell ordinary border cell nodal Hensen cell Purkinje fiber Supporting cell of vestibular apparatus Smooth muscle cells (various) Supporting cell of taste bud (type I taste bud cell) myoepithelial cells Supporting cell of olfactory epithelium of iris 25 Schwann cell of exocrine glands satellite cell (encapsulating peripheral nerve cell bodies) Cells of Blood and Immune System enteric glial cell Neurons and Glial Cells of Central Nervous System red blood cell megakaryocyte neurons (huge variety of types-Still poorly classified) macrophages and related cells 30 glial cells monocyte astrocyte (various) connective-tissue macrophage (various) oligodendrocyte Langerhans cell (in epidermis) Lens Cells Osteoclast (in bone) dendritic cell (in lymphoid tissues) anterior lens epithelial cell microglial cell (in central nervous system) lens fiber (crystallin-containing cell) neutrophil 35 Pigment Cells eosinophil basophil melanocyte mast cell retinal pigmented epithelial cell T lymphocyte Germ Cells helper T cell Suppressor T cell 40 Oogonium oocyte killer T cell spermatocyte B lymphocyte spermatogonium (stem cell for spermatocyte) IgM Nurse Cells IgG IgA ovarian follicle cell IgE 45 Sertoli cell (in testis) killer cell thymus epithelial cell stem cells and committed progenitors for the blood and Exocrine secretory epithelial cells immune system (various) Sensory Transducers Salivary gland mucous cell (polysaccharide-rich secretion) Salivary gland number 1 (glycoprotein enzyme-rich secretion) photoreceptors 50 Von Ebner's gland cell in tongue (washes taste buds) rod Mammary gland cell (milk secretion) COile:S Lacrimal gland cell (tear Secretion) blue sensitive Ceruminous gland cell in ear (earwax Secretion) green sensitive Eccrine Sweat gland dark cell (glycoprotein secretion) red sensitive Eccrine Sweat gland clear cell (Small molecule secretion) hearing 55 Apocrine Sweat gland cell (odoriferous secretion, sex-hormone inner hair cell of organ of Corti sensitive) Outer hair cell of organ of Corti Gland of Moll cell in eyelid (specialized sweat gland) acceleration and gravity Sebaceous gland cell (lipid-rich sebum Secretion) type I hair cell of vestibular apparatus of ear Bowman's gland cell in nose (washes olfactory epithelium) type II hair cell of vestibular apparatus of ear Brunner's gland cell in duodenum (enzymes and alkaline taste mucus) type II taste bud cell 60 Seminal vesicle cell (secretes Seminal fluid components, Smell including fructose for Swimming sperm) olfactory neuron Prostate gland cell (secretes Seminal fluid components) basal cell of olfactory epithelium (stem cell for olfactory neurons) Bulbourethral gland cell (mucus secretion) blood pH Bartholin's gland cell (vaginal lubricant secretion) carotid body cell Gland of Littre cell (mucus secretion) type I 65 Uterus endometrium cell (carbohydrate secretion) type II Isolated goblet cell of respiratory and digestive tracts (mucus US 9,714,897 B2 47 48 TABLE 6-continued TABLE 6-continued Secretion) Nervous system Stomach lining mucous cell (mucus Secretion) Gastric gland zymogenic cell (pepsinogen secretion) There are nerve cells, also known as neurons, present in our human Gastric gland oxyntic cell (hydrochloric acid secretion) body. They are branched out. These cells make upnervous tissue. Pancreatic acinar cell (bicarbonate and digestive enzyme A neuron consists of a cell body with a nucleus and cytoplasm, Secretion) from which long thin hair-like parts arise. Paneth cell of Small intestine (lysozyme secretion) Sensory transducer cells Type II pneumocyte of lung (Surfactant Secretion) Clara cell of lung Auditory inner hair cell of organ of Corti Hormone secreting cells 10 Auditory outer hair cell of organ of Corti Basal cell of olfactory epithelium (stem cell for olfactory Anterior pituitary cells neurons) Somatotropes Cold-sensitive primary sensory neurons Lactotropes Heat-sensitive primary sensory neurons Thyrotropes Merkel cell of epidermis (touch sensor) Gonadotropes 15 Olfactory receptor neuron Corticotropes Pain-sensitive primary sensory neurons (various types) Intermediate pituitary cell, Secreting melanocyte-stimulating Photoreceptor cells of retina in eye: hormone Photoreceptor rod cells Magnocellular neurosecretory cells Photoreceptor blue-sensitive cone cell of eye Secreting oxytocin Photoreceptor green-sensitive cone cell of eye Secreting vasopressin Photoreceptor red-sensitive cone cell of eye Gut and respiratory tract cells Proprioceptive primary sensory neurons (various types) Secreting serotonin Touch-sensitive primary sensory neurons (various types) Secreting endorphin Type I carotid body cell (blood pH sensor) Secreting somatostatin Type II carotid body cell (blood pH sensor) Secreting gastrin Type I hair cell of vestibular system of ear (acceleration and Secreting secretin gravity) Secreting cholecystokinin 25 Type II hair cell of vestibular system of ear (acceleration and Secreting insulin gravity) Secreting glucagon Type I taste bud cell Secreting bombesin Autonomic neuron cells Thyroid gland cells thyroid epithelial cell Cholinergic neural cell parafollicular cell 30 Adrenergic neural cell Parathyroid gland cells Peptidergic neural cell Parathyroid chief cell Sense organ and peripheral neuron Supporting cells Oxyphil cell Adrenal gland cells Inner pillar cell of organ of Corti chromaffin cells Outer pillar cell of organ of Corti Secreting steroid 35 Inner phalangeal cell of organ of Corti hormones (mineralcorticoids and gluco Outer phalangeal cell of organ of Corti corticoids) Border cell of organ of Corti Leydig cell of testes secreting testosterone Hensen cell of organ of Corti Theca interna cell of ovarian follicle Secreting estrogen Vestibular apparatus Supporting cell Corpus luteum cell of ruptured ovarian follicle Taste bud Supporting cell Olfactory epithelium supporting cell Secreting progesterone 40 Granulosa lutein cells Schwann cell Theca lutein cells Satellite glial cell (encapsulating peripheral nerve cell bodies) Juxtaglomerular cell (renin secretion) Enteric glial cell Macula densa cell of kidney Central nervous system neurons and glial cells Peripolar cell of kidney Astrocyte (various types) Mesangial cell of kidney 45 Neuron cells (large variety of types, still poorly classified) Derived primarily from ectoderm Oligodendrocyte Integumentary system Spindle neuron Keratinizing epithelial cells Lens cells Epidermal keratinocyte (differentiating epidermal cell) Anterior lens epithelial cell Epidermal basal cell (stem cell) 50 Crystallin-containing lens fiber cell Keratinocyte of fingernails and toenails Derived primarily from mesoderm Nail bed basal cell (stem cell) Metabolism and storage cells Medullary hair shaft cell Cortical hair shaft cell Hepatocyte (liver cell) Cuticular hair shaft cell Adipocytes: Cuticular hair root sheath cell 55 White fat cell Brown fat cell Hair root sheath cell of Huxley's layer Liver lipocyte Hair root sheath cell of Henle’s layer Barrier function cells (lung, gut, exocrine External hair root sheath cell glands and urogenital tract) Hair matrix cell (stem cell) Kidney Wet stratified barrier epithelial cells 60 Kidney parietal cell Surface epithelial cell of stratified squamous Kidney glomerulus podocyte epithelium of cornea, tongue, oral cavity, esophagus, anal Kidney proximal tubule brush border cell canal, distallurethra and vagina Loop of Henle thin segment cell basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, Kidney distal tubule cell esophagus, anal canal, distal urethra and vagina Kidney collecting duct celldisambiguation needed Urinary epithelium cell (lining urinary bladder and urinary 65 Type I pneumocyte (lining air space of lung cell) ducts) Pancreatic duct cell (centroacinar cell) US 9,714,897 B2 49 50 TABLE 6-continued TABLE 6-continued Nonstriated duct cell (of Sweat gland, salivary gland, mammary Germ cells gland, etc.) principal cell Oogonium Oocyte Intercalated cell Spermatid Duct cell (of Seminal vesicle, prostate gland, etc.) Spermatocyte intestinal brush border cell (with microvilli) Spermatogonium cell (stem cell for spermatocyte) Exocrine gland striated duct cell Spermatozoon Gall bladder epithelial cell Nurse cells Ductulus efferens nonciliated cell Epididymal principal cell 10 Ovarian follicle cell Epididymal basal cell Sertoli cell (in testis) Extracellular matrix cells Thymus epithelial cell Interstitial cells Ameloblast epithelial cell (tooth enamel secretion) Planum semilunatum epithelial cell of vestibular system of ear (proteoglycan secretion) Interstitial kidney cells Organ of Corti interdental epithelial cell (secreting tectorial 15 membrane covering hair cells) Loose connective tissue fibroblasts Corneal fibroblasts (corneal keratocytes) TABLE 7 Tendon fibroblasts Bone marrow reticular tissue fibroblasts B Cell maturation markers for use with the Other nonepithelial fibroblasts hydrogel particles described herein. Pericyte Nucleus pulposus cell of intervertebral disc B-cell Cementoblasticementocyte (tooth root bonelike ewan cell type Cell Surface marker(s) Secretion) Odontoblastodontocyte (tooth dentin secretion) Pro-B CD19, CD2O, CD34, CD38, CD45R Hyaline cartilage chondrocyte 25 Pre-B CD19, CD2O, CD38, CD45R Fibrocartilage chondrocyte Immature B CD19, CD2O, CD40, CD45R, IgM Elastic cartilage chondrocyte Tr-B CD10, CD19, CD2O, CD24, CD28 Osteoblastosteocyte Naive-B CD19, CD2O, CD23, CD40, CD150 (SLAM), Ig|D, IgM B-1 CD19, CD2O, CD27, IgM Osteoprogenitor cell (stem cell of Osteoblasts) Memory B Hyalocyte of vitreous body of eye CD19, CD2O, CD28, CD40, IgA, IgG 30 Plasma Cell CD19, CD28, CD31, CD38, CD40, CD95 (FAS), CD184 Stellate cell of perilymphatic space of ear (CXCR4) Hepatic stellate cell (Ito cell) Pancreatic stelle cell Contractile cells TABLE 8 skeletal muscle Cell 35 Red skeletal muscle cell (slow) Cell surface markers for use with the White skeletal muscle cell (fast) hydrogel particles described herein. Intermediate skeletal muscle cell nuclear bag cell of muscle spindle 14-3 -3 - ? nuclear chain cell of muscle spindle 14-3 -3 II. Satellite cell (stem cell) 40 14-3 -3 I' Heart muscle cells 14-3-3 I, Ordinary heart muscle cell 14-3-3 If Nodal heart muscle cell 15-Lipoxygenase 1 Purkinje fiber cell 160 kD Neurofilament Medium Smooth muscle cell (various types) 200 kD Neurofilament Heavy Myoepithelial cell of iris 45 3G 11 Sialoganglioside antigen Myoepithelial cell of exocrine glands 4E-BP1 Blood and immune system cells 4E-BP1. Phospho (Thr37/46) 5-Methylcytidine Erythrocyte (red blood cell) 5HT3A receptor Megakaryocyte (platelet pecursor) ST4 Monocyte (white blood cell) 50 68 kDa Neurofilament Light Connective tissue macrophage (various types) 7 Epidermal Langerhans cell 70 kD Neurofilament Light Osteoclast (in bone) Dendritic cell (in lymphoid tissues) Microglial cell (in central nervous system) Neutrophil granulocyte 55 Eosinophil granulocyte Basophil granulocyte Hybridoma cell Mast cell Helper T cell Suppressor T cell 60 Cytotoxic T cell Natural Killer T cell B cell Natural killer cell Reticulocyte ACAA1 Stem cells and committed progenitors for the blood and immune 65 ACADM system (various types) ACAT2 US 9,714,897 B2 51 52 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. ACBD3 ACD LDOB ACE2 dolase B Acetyl Coenzyme A Carboxylase exa Fluor 405, Cascade Blue Acetyl Coenzyme A Carboxylase It exa Fluor 488 Acetyl Coenzyme A Synthetase Acetylated Lysine 10 ix AChRIt lergin1 AChRI ha 1 Antitrypsin AChRI ha 1 Catenin Aconitase2 ha 1 Sodium Potassium ATPase ACOT12 ha2 Catenin ACSA2 15 ha 2 Macroglobulin ACSF2 ha. Actin 1 ACSMS ha. Actin 2 Act1 ha. Actinin Activation molecule 8 (B cells) ha. Actinin 2 Activin A Receptor Type IB ha. Actinin 3 Activin A Receptor Type IIB ha. Actinin 4 ACTN3 ha. Adaptin ACY1 ha. Adducin ACY3 ha B Crystallin ADA ha Fodrin ADAM12 ha Internexin ADE.2 ha. Synuclein Adenosine A1 Receptor 25 Adenosine A2aR AMACR Adenovirus Aminopeptidase P Adenovirus Fiber monomer and trimer AML.1 Adenovirus hexon protein Amphiphysin Adenylate Kinase 1 AMPKIt AdenyloSuccinate Lyase 30 AMPKI + 1 ADFP AMPKI + 2 ADH1B AMPKI1 ADH6 AMPKP1 ADH7 AmyloidI42 ADI1 ANAPC2 Adiponectin AND1 Adiponectin Receptor 2 35 Androgen Receptor Adipose Triglyceride Lipase Angiotensin I ADP Ribosylation Factor Angiotensin II Receptor 2 ADP-ribosyltransferase 2.2 gene Angiotensin III Adrenodoxin ANKRDS3 AF10 Annexin IV AFAP1 40 Annexin V AFP ANP AG2 Anti-Kudoa thrysites AGAP1 Anti-T. brucei procyclin (GPEET) AGPATS Anti-T. brucei procyclin (phosphorylated GPEET) GR2 Antiglobulin (Coombs) 45 Antithrombin III CDA P2 it P2 i + 2 P2 3 Aiolos PL1 50 PAF1 RE PBB3 PC PC-1 KS PC-10 kt PC-11 kt (pS473) 55 PC-2 kt (pT308) PC-3 kt1 PC-5 PC-7 PC-8 bumin PE1 cohol Dehydrogenase PG12 dehyde Reductase 60 LDH1A1 LDH1L1 LDH2 PMAP LDH3A1 bo-2.7 LDH3A2 po-2.7 (7 A6) LDHSA1 65 boE LDH6A1 boE4 US 9,714,897 B2 53 54 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. POER2 BAG1 polipoprotein AI BAG2 polipoprotein AII BAG3 polipoprotein AIV BAG4 polipoprotein B BAIAP2 polipoprotein CIII BAK polipoprotein D 10 BAMBI polipoprotein E BAP31 polipoprotein F BAP37 polipoprotein H basal cell Cytokeratin polipoprotein J Basophils polipoprotein L1 Bassoon polipoprotein M 15 BATF otic neutrophils Bax BCAR1 borin 1 BCAR2 borin 5 BCKD complex E2 subunit Bcl-10 5 Bcl-2 GAP1 Bcl-2 (pS70) RP1 Bcl-2 like 12 onaute-1 Bcl-2 like 2 H Bcl-22 HGAP2S HGAP4 Bcl-2It L11 25 Bcl-3 Bcl-6 PC5 Artemis Bcl-XSL Aryl hydrocarbon Receptor BCR BCSC1 30 BDKRB2 ASGPR BDNF Asialo-GM1 Beclin1 ASK1 Bestrophin 3 Asparagine synthetase beta 2 Adrenoreceptor Ataxin 1 35 Beta 3 Adrenergic Receptor ATF1 beta 3 Sodium Potassium ATPase ATF2 beta Actin beta Arrestin 1 beta Arrestin 2 ATIC beta Catenin Atlantic Salmon Ig beta Catenin (npaa 27-37) ATM 40 beta Catenin (npaa 35-50) ATP citrate lyase beta Catenin (pS45) beta Dystroglycan ATPSA beta galactosidase beta galactosidase fusion proteins ATP5 beta Synuclein ATPSO 45 beta2 Microglobulin ATP6VOD1 BHMT Bid ATPB Biglycan ATRIP Bilirubin Oxidase Aurora A Bim Aurora A Phospho (Thr288) 50 BimL Aurora B BIN1 Aurora B Phospho (Thr232) AVEN iotin Avian Influenza A Neuraminidase P Avidin Axin 2 55 imp-1 Axl B and Activated T Cells LNK B Cell LNK (p Y84) B Cell Subset ood Group A. Antigen B cells (pan reactive) ood Group AB Antigen B lymphocytes antibody UCH-B1 ood Group B Antigen b-Endorphin 60 ood Group H ab Antigen B-Raf Phospho (Thr598/Ser601) ood Group H ab Antigenn Antigen B18R ood Group H inhibitor ood Group Lewis a BACE1 ood Group M Antigen BACE2 ood Group N Antigen BACH1 65 ooms Syndrome Protein Blm baculovirus envelope gp64 protein BM1 US 9,714,897 B2 55 56 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. BMAL1 5 CAB39 BMI1 CACNA1S Bmk CACNA2 BMP15 CACNG1 BMP4 CAD BMP7 Cadherin 1 BMPR1A 10 Cadherin 10 BMPR2 Cadherin 11 BMX Cadherin 7 bMyc Cadherin 8 BNIP2 Cadherin 9 BNIP3 Cadherin E BNIP3L 15 Cadherin H BOB1 Cadherin K BORA Cadherin P Borealin Cadherin R Borrelia burgdorferi CAKC Terminus BPI CAKN Terminus BRaf CAK Phospho (Ser164/Thr170) BRCA1 2O Calbindin BRCC36 Calcineurin A BRD3 Calcitonin Receptor Bird Calcium Sensing Receptor BRF1 Caldesmon BRG1 Calgranulin A BRN3A 25 Calgranulin B Btk Calmodulin Btk (pY551)/Itk (pY511) Calnexin - ER membrane marker BTLN-2 Calpain 1 BTN1A1 Calpain 2 Bu1 Calpain 9 Bu1a 30 Calpain S1 (Small subunit) Bu1a Bulb Calpastatin Bulb Calponin BubR1 Calreticulin Bulb Calretinin Butyrylcholinesterase Calsequestrin 2 C peptide 35 CaMKI C reactive protein CaMKII C/EBP12 CaMKII Phospho (Thr286) C1. Inhibitor CaMKIII C16orf72 CaMKII: C1 orf50 CAMLG C1Q 40 cAMP Protein Kinase Catalytic subunit C1QA cAMP Protein Kinase Catalytic subunit I+ C1QB Cannabinoid Receptor I C1QC Cannabinoid Receptor II C1QG CAP-G2 C1r CAP18 C1s 45 CAP2 C20orf50 CAP3 C20orfA3 Carbonic Anhydrase I C21orf56 Carbonic Anhydrase IX C21orf59 Carboxylesterase 1 C2OrfA3 Carboxypeptidase A1 C3 50 Carboxypeptidase A2 C3aR CARD11 C3b CARD8 C3c. CARD9 C3d Cardiac Troponin T C4 CARKL C4 binding protein 55 CARM1 - C4b Casein Kinase 1 It C4c Casein Kinase 1 12 C4d Casein Kinase 2 I C4OrfA2 Caspase 1 C5 Caspase 10 C5aR1 Caspase 11 CSL2 60 Caspase 12 C6 Caspase 2 C6orf64 Caspase 2L C8A.B.G. Caspase 3 C9 Caspase 4 C9orfA1 Caspase 5 CA12S 65 Caspase 6 CA19.9 Caspase 7

US 9,714,897 B2 63 64 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. CD79b 5 CHD4 CD8 Chemerin CD8O CHIPS, C-terminus CD81 CHIPS, N-terminus CD82 Chk1 CD83 Chk2 CD84 10 Chondroitin Sulfate CD85 CHOP CD85a Chromogramin C CD85d CT1 CD85g chTOG CD8Sh cIAP1 CD85 15 cIAP2 CD8Sk CIAS1 CD86 CIDEA CD87 CIP4 CD88 CISD1 CD89 CITED1 CD8It CITED2 CD8+ 1 2O cJun CD8I 2 cJun Phospho (Tyr91/TyrS3) CD82 CKIII: CD9 CKMT2 CD90.1 CLASP1 CD90.2 Clathrin CD90.9 25 Claudin-1 CD91 Claudin-10 CD91It Claudin-15 CD912 Claudin-16 CD93 Claudin-18 (C-term) CD94 Claudin-18 (Mid) CD95 30 Claudin-4 CD96 Claudin-5 CD97 Claudin-8 CD98 CLAW-H CD98hc CLEC12A CD99 CLEC1B CD99R 35 CLEC4A Coc-123 CLEC4M Cdc-2 (p34) CLEC9A Cdc-25A Phosph (Ser17) CLIP Coc-25C CLOCK Coc-37 Cliostridium bointinum Toxin B Coc-4SL CLPP Colc-6 40 cMaf CDC-7 cMet Cck1 CMKLR1 Cok2 CMRF44 Codka. CMRFS6 CokS cMyb Cokó 45 cMyc Cdkf CNDP2 Cok9 CNTFRIE CokA1 COASY CokN2A Coatomer I CdkN3 Cofilin CDT1 50 Colec12 CDX2 Collagen I CEACAM19 Collagen I/III CEACAM2O Collagen II CEACAM7 Collagen III CEBPIE Collagen IV CEBP12 55 Collagen V CEND1 Collagen VI CENPA Collagen VII CENPE COMMD1 CENPF Complement Factor B CENPH Complex I Immunocapture Centrin 2 Conjugated Choline Glutaric acid CFAH 60 Connexin 26 cFos Connexin 30 CFTR Connexin 30.2 CGBS Connexin 30.3 cGK1 Connexin 32 CH2 Connexin 36 CHCHDS 65 Connexin 37 CHD3 Connexin 37 (C-term) US 9,714,897 B2 65 66 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. Connexin 37 (Mid) CXCL9 Connexin 39 CXCR7 Connexin 39 (Mid) CXorf26 Connexin 40 (C-term) Cyanine Connexin 40 (Mid) CYB5R2 Connexin 43 CYB5R3 Connexin 45 10 Cyclin A Connexin 45 (C-term) Cyclin A2 Connexin 46 Cyclin B1 Connexin 47 Cyclin B2 Connexin 57 (C-term) Cyclin D1 Connexin 57 (Mid) Cyclin D2 Contactin 2 15 Cyclin D3 COPS3 Cyclin E Coronavirus Cyclin E2 Coronin 1A Cyclin H Coronin 1B Cyclins D1/D2/D3 Cortactin Cyclophilin 40 Cortical Thymocytes CYLD COXI CysLT1 COX IIII Cystatin C COX II Cystatin S COX IV Cytochrome B245 heavy chain COXVA Cytochrome B245 light chain COX VIA1 Cytochrome c Coxsackie Adenovirus Receptor 25 Cytochrome P450 17 A1 PF Cytochrome P450 19A1 PI17It Cytochrome P450 1A2 bn10 Cytochrome P450 2A6 PO Cytochrome P450 2B6 PS1 Cytochrome P450 2C9 PT2 30 Cytochrome P450 2J2 RABP1 Cytochrome P450 3A4 RABP2 Cytochrome P450 3A5 RALBP Cytochrome P450 Reductase reatine Kinase BB Cytokeratin reatine Kinase MM Cytokeratin (acidic) REB 35 Cytokeratin (basic) REB Phospho (Ser133) Cytokeratin (Pan-reactive) cRel Cytokeratin 1 ripto1 Cytokeratin 10 RISP3 Cytokeratin 10/13 rk p38 Cytokeratin 13 rk Cytokeratin 14 rkL (pY207) 40 Cytokeratin 14/15/16/19 ROT Cytokeratin 15 RRY Cytokeratin 16 RTAM Cytokeratin 17 RTC3 Cytokeratin 18 RY2 Cytokeratin 19 ry ptochrome I 45 Cytokeratin 2 ryptosporidium Cytokeratin 20 ryptosporidium Parvum Cytokeratin 4 RYZL.1 Cytokeratin 4/5/6/8/10/13/18 CSK Cytokeratin 40 CSK Binding Protein Cytokeratin 5 CSPS 50 Cytokeratin 5/6/18 cSrc Cytokeratin 5/8 CST2 Cytokeratin 6 CTDSP1 Cytokeratin 6a CTNNA3 Cytokeratin 7 CTNNBL1 Cytokeratin 7/17 Cullin 1 55 Cytokeratin 8 Cullin 2 Cytokeratin 8/18/19 Cullin 3 Cullin 4A DAB2 Cullin 4AB DACH1 Cullin 4B DANDS Cutaneous Lymphocyte Antigen DAP1 CUTL1 60 DAP12 CX3CL1 DAPK1 CX3CR1 DAPK2 CXCL1 DARPP32 CXCL10 Daxx CXCL12It DAZL CXCL1212 65 DBC1 CXCL13 DCAMKL1 US 9,714,897 B2 67 68 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. DCC DCIR2 DUSP23 DCLRE1B DUSP27 DUSP3 DcR3 DUSP5 DCTN2 DUSP6 DcTRAIL-R1 10 DUX4 DcTRAIL-R2 DYKDDDDK Epitope Tag Dynamin DDB1 Dynamin1 DDDDK tag Dynamitin DDX3 Dynein light chain 2 DDX4 15 Dysbindin DDXSO Dysferlin DECR1 Dystrobrevin It Dectin1 Dystrobrevin I? Dectin2 Dystroglycan Phospho (Tyr893) DEF8 E. Coii OE Defensin I + 1 DELETE delta 1 Catenin Delta like protein 1 E4BP4 Delta like protein 4 EaS2-68 peptide bound to I-A Delta Opioid Receptor EaS2-68 peptide bound to the I-A DeltaC EAAT1 DeltaD 25 Early B Lineage Dendritic Cell Marker Deoxycytidine kinase BI3 Desmin BPSO Desmoglein 2 Desmogleinl CH1 Desmoplakin 30 CRG4 Destrin DA Dextran DA-A2R DGKA Dicer DG2 DISC1 (C-term) DG3 DISC1 (Mid) 35 Dishevelled 3 EA1 Disialoganglioside GD2 Disialoganglioside GD3 EF2 Dkk1 EN DLC8 FEMP1 DLK1 40 FEMP2 5 DM-GRASP Eg5 Phospho (Thr927) EGF DNA-PKCS EGF Receptor DNA-PKcs Phospho (Thr2609) EGF Receptor (pY1173) DNAI1 45 EGF Receptor (pY845) DNAJA2 EGF Receptor (pY992) DNATB2 EGR1 DNAJC3 EGR2 DNAPK EHD1

Dnmt1 50 Dnimt3b DNP DOK2 DOK7 Dopamine Receptor D1 F3D (p66) Dopamine Receptor D3 Dopamine Receptor D5 55 Dopamine 2 Hydroxylase Doublecortin F3I (p36) DP1 DPH2 DPP10 DPP3 60 DPP9 F4E (pS209) Dppa4 DPYD eIFSA DR3 DRAK1 Elastase DRAK2 65 Drebrin Elk1 (pS383) US 9,714,897 B2 69 70 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. ELK3 5 F-actin Elongin B F10A1 Elongin C F4.80 EMAP II FAA4 Embigin FABP4 EMG1 Factor I Emil 10 Factor IX EMR3 Factor VIII.v.WF (delete) EMSY Factor XIIIa Ena Vasp-like FADD EndoG FAHD2A EndoGlyX-1 FAK Endomlucin 15 FAK (pS910) Endothelial Cells FAM119A Endothelial Lipase FAM17SA Endothelial Venule Marker FAM84B Endothelium FAM91A1 Engrailed1 EANCC ENO1 EANCD2 Enolase1 2O Fanconi anemia D2 Phospho (Ser222) eNOS FAP eNOS (pS1177) Fascin Entpd2 FBP1 Eomes FBXO21 Eos FBXO31 Epac1 25 FBXO42 Eph Receptor A1 FBXO43 Eph Receptor A2 Fc Receptor Binding Inhibitor Eph Receptor A4 Fc receptor IgA + IgM Eph Receptor B4 FCR Eph Receptor B6 FcRL6 Ephrin A2 30 FcRLA Ephrin A3 FciuRI EPHX2 FDC EPM2AIP1 FDFT1 EPOR FDPS EPS15R FE65 Epsin 1 35 FeLV p27 Epsin 2 FEN1 E FER E Ferritin Heavy Chain E Ferritin Light Chain E Ferritin, mitochondrial E FES E 40 Fetal Hemoglobin E FGF acidic ERK1 FGF basic ERK1/2 (pT185 pY187) FGF21 ERK 1/2 (pT202 pY204) FGFR1 ERK1, ERK2 FGFR2 ERK2 45 FGR. ERKS FH ERMAP FHL1 ERp29 Fibrillarin ERp72 Fibrillin Erythroid Cells Fibrinogen Erzin Radixin Moesin 50 Fibrinogen i it chain ERI + Phospho(Ser167) Fibrinogen I chain ESAM Fibrinopeptide A Estrogen Inducible Protein pS2 Fibrinopeptide B Estrogen Receptor - Fibroblast activation protein It Estrogen Receptor It Fibroblast Surface Protein Estrogen Receptor I? 55 Fibroblasts/Epithelial cells Estrogen Related Receptor alpha Fibronectin ETAR Fibronectin Receptor Ethenoadenosine FibulinS ETS1 Ficolin B EVI2A Filaggrin EVI2B Filamin A EWSR1 60 FITC EXD1 FITC/Oregon Green EXOSC3 FIV EXOSC7 FIV gp120 EYA2 FIV gp95 EZH1.2 FIV p24 Ezrin 65 FIV p24gag Ezrin (pY353) FKBP12 US 9,714,897 B2 71 72 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. FKBP4 5 GATA1 FKBP6 GATA2 FKBPL GATA3 FLC GATA4 Flightless 1 GATM FLIP GBA3 Flt3L 10 GBE1 Fluorescent Protein GBP1 FLV gp70 GBP2 FLYWCH2 GBP5 MLP Receptor GCDFP15 FMRP 15 GCDH FNTA GCK1 FNTB GCLM Follicular Dendritic Cells GCN2 Fos GCN5 FOXA1 GCTM2 FOXA2 GDAP1L1 FOXC2 2O GDF15 FOXD3 Gelsolin FOX1 Gemin1 FOX1 Gephyrin FOXM1 GFAP FOXO1 GFP FOXO3A 25 GILZ FOXP1 GIMAP4 FOXP3 GIPR FPRL1 GIT2 FR4 GITRL Fra GLAST Fragilis 30 Gli1 FRAT1 Glial Fibrillary Acidic Protein Frataxin Glicentin Frequenin GLIPR1L1 Frizzled-1 Glucagon FSHIE Glucocorticoid Receptor FSH2 35 Glucocorticoid Receptor alpha FUK Glucose 1 Dehydrogenase FUS Glucose 6 Phosphate Isomerase FXYD3 GLUH1 FYB GLUT1 Fyn GLUT2 Fyn (pY528), c-Src (py530) GLUT4 Fyn-Related Kinase 40 GLUT5 FZR1 Glutamate receptor 2 G-CSF Glutamate receptor 2/3 G3BP Glutamate receptor 3 G6PD Glutamate receptor 4 GAB1 Glutaminase GAB2 45 Glutamine Synthetase GABAB Receptor 2 Glutaredoxin 2 GABARAP Glutathione NEM GAD6S Glutathione NEW GAD67 Glutathione Peroxidase 1 GADD34 Glutathione Peroxidase 4 Galacto-cerebroside 50 Glutathione Reductase Galactocerebroside Glutathione S Transferase I, 2 Galectin 1 Glutathione S Transferase I-1 Galectin 10 Glutathione S Transferase I/4 Galectin 3 Glutathione Synthetase Galectin 4 Glycogen synthase 1 Galectin 7 55 Glycoprotein DX Galectin 8 Glycoprotein VI Galectin 9 GM-CSF gamma Synuclein GM130 Ganglioside GD2 GM3.2 Ganglioside GD3 GNB2 Ganglioside GM1 GNB2L1 Gankyrin 60 GNLY GAP GNMT GAP43 GnRHR GAPDH Golgi Protein (58K) GARP Golgi Zone GAS2 GOLM1 GAS7 65 GOLPH2 GAT2 GOSR1 US 9,714,897 B2 73 74 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein.

l CGI24

DAC1 DAC10 DAC2 DAC3 10 DAC4 DAC6 DAC9

DHD2

15 DLBP EC1 EF1 elios ematopoiesis related Macrophage ematopoietic Lineage Cocktail ematopoietic Progenitor Cell emoglobin emoglobin F Granulysin emoglobin Subunit I+ Granzyme A epatitis B Virus Granzyme B epatitis B Virus Core Antigen Granzyme K 25 epatitis B Virus E Antigen GRAP2 epatitis B Virus Surface Antigen (Ad/Ay) GRASP1 epatitis C Virus GRASP65 epatitis C Virus Core Antigen GRB2 epatitis C Virus NS4 GRB7 epsin GRHPR 30 ER3 GRIM19 ER4 GRK1 es1 GRK2 exokinase GRK3 exokinase1 GRKS exokinase2 GRK6 35 FE1 Growth hormone receptor GRP170 GFA Inhibitor 1 GRP94 HEX HV8 GPCR IBCH ID1 40 GSPT2 GST GST Epitope Tag INT1 GSTA4 IP2 GTF2D1 GTPase HRAS 45 ippocalcin GTPBP4. istamine H3 Receptor Guanylate kinase istocytes H-2 istone H1 H-2.m31 istone H1.0 istone H2A 50 istone H2B istone H2B type 1B istone H3 istone H3 Phospho (Ser10) H2AX istone H3 Phospho (Ser28) H2AX Phospho (Ser139) istone H3.3 H2A1 istone H4 55 IV1 Core Antigen HA tag IV1 p17 HADHA IV1 p24 HADHAHADHB HADHB IV1 tat HADHSC HAND1 60 LA Class I HAO1 LA-2Kb. 2Db Haptoglobin LA-2kb 2Dd HARS LA-A HARS2 LA-AB,C LA-A1, A11 A26 hCGIt 65 LA-A1 A36 LA-A10/A11

US 9,714,897 B2 79 80 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. KDEL

LK1 Ki-67 ImmunofluorescenceN-I KIF22 MP3 importin9 influenza A Virus M2 Protein 10 KIFA3 influenza B Virus Nucleoprotein Kindlin2 NG1 Kinetoplastid Membrane Protein 11 (KMP-1)) NG2 R-2.1 NG3 R-2D (pan CD158) NG4 LF4 (nhibin it 15 NOS insulin insulin Degrading Enzyme (IDE) insulin Receptor R integrin I 4/27 integrin It 9/11 KOR-SA3544 integrin It VI's integrin It VI26 Ksp37 integrin 121 Phospho (Tyr783) KSR1 integrin 121 Phospho (Tyr795) Ku70 integrin Is 25 Ku70.80 integrin I6 integrin I’7 Kudoa Thyrsites intercalated DNA Kunitz Protease Inhibitor intra Acrosomal Protein Kv4.2 intra-Acrosomal Proteins LS-MAG nvariant NK T 30 Labeling Check Reagent P10 Lactate Dehydrogenase Lactate Dehydrogenase B RAK1 Lambda RAK3 Lamin A RAK4 Lamin AC RE1 35 Lamin B Receptor Lamin B1 Lamin B2 Lamin C Laminin Laminin 5 Laminin Receptor RF7 (pS477 pS479) 40 Laminin I1 LAMP2a LAMP2b RS1 LAT RS1 (pY896) LAT (pY171) RS2 LAT (pY226) RS4 45 SG15 SG20 SL1 LCAT sthmin1 Lck TCH Lck (pY505) integrin i + 7 50 LDH1 BC TPR1 LDL (MDA oxidized) agged2 LDLR AK2 LEF1 AK3 Leishmania LPG (repeat epitope) AM2 55 Leishmania Major Surface Protease (GP-63) AML LEKTI apanese encephalitis virus NS1 glycoprotein Leukemia Inhibitory Factor JNK Leukotriene A4 hydrolase JNK Phospho (Thr183/Tyr185) Leukotriene B4 Receptor JNK1 TNK2, NK3 LHX3 JNK2 LI-Cadherin Junctional Adhesion Molecule C 60 LIF Junctophilin-1 (C-term) DNA Ligase I Junctophilin-1 (Mid) DNA Ligase III Junctophilin-2 (C-term) LIM kinase 2 Junctophilin-3 (C-term) LIME1 KAP1 LIMK1 KATNA1 65 LIMS1 Lin28 US 9,714,897 B2 81 82 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. Lineage Cocktail MAP2K1IP1 Lipin 1 MAP3K8 LIS1 MAP4 Phospho (Ser768) Liver Carboxylesterase 1 MAP4K1 LKB1 MAP4K4 LMO2 MAPK12 LOX 10 MA K 6 LOX1 MA KAP Kinase 2 LRPS, 6 MA KAP Kinase 2 Phospho (Thr334) MA CKS LRPAP1 MA CO LSD1 M arginal Zone B Cells LSP1 15 MA K2 LSS MARK3 LTIt MART1 Luciferase Mast Cell Mast Cell Protease 11 Ly-108 mature macrophage marker MBD MBD2 MBL MCL MCM2 MCM3 MCM4 25 MCMS MCM6 MCM7

30

35 EK1 EK1 (p298) EK1 (pS218)/MEK2 (pS222) Ly-77 EK1/2 (pS222) Lymphotoxin 2 EK2 Lymphotoxin I Receptor EK3 Lyn 40 LYRIC EKS Lysophospholipase 1 Lysosomal acid lipase Lysozome Lysozyme Lyve1 45 M-CSF M13 Bacteriophage Coat Protein g8p e M13 Bacteriophage Protein MAA Mac-2BP macroH2A.1 50 Macrophage Macrophage Activator Macrophage galactose lectin Macrophage, Granulocyte Mesothelin Macrophages/Monocytes Metallothionein MAD2 55 MadCAM1 MADD MADH7 MHC Class I MAFB MHC Class I (H-2Db) MAG MHC Class I (H-2Dd) MAGE-A MHC Class I (H-2Dk) MAGE1 60 MHC Class I (H-2Dq/Lc) MAIR2 MHC Class I (H-2Kb) MAIR4 MHC Class I (H-2Kb/Db) MALT1 MHC Class I (H-2Kb/Dd) Mammaglobin A MHC Class I (H-2Kda3 domain) MAP1LC3A MHC Class I (H-2Kd) MAP2 65 MHC Class I (H-2Kd/Dol) MAP2B MHC Class I (H-2Kd/Dal/q/u/v) US 9,714,897 B2 83 84 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. MHC Class I (H-2Kk) 5 MRP14 MHC Class I (H-2Kq) MRP2 MHC Class I (H-2Ks) MRP3 MHC Class I (H-2Ld) MRP4 MHC Class I (H-2Ld/Db) MRP5 MHC Class Ib (H2-M3) MRP6 MHC Class 10 MRP8 MHC Class II (DQ) MRP8,14 MHC Class II (DR) MSC (W8B2) MHC Class II (I-A) MSC (W3D5) MHC Class II (I-A/E) MSC (W5C5) MHC Class II (I-Ab) MSC (W7C6) MHC Class II (I-Ab/Ad) 15 MSCNPC MHC Class II (I-Ab/As) MSH2 MHC Class II (I-Ad) MSH6 MHC Class II (I-Ak) MSI2EH MHC Class II (I-Ak/Ad Ab/Aq/Ar) MSK1 MHC Class II (I-Ak/As) MST1 MHC Class II (I-Ap) MST1 MST2 MHC Class II (I-Aq) 2O MST3 MHC Class II (I-E) MST4 MHC Class II (I-EI) MST4, MST3:STK2S MHC Class II (RT1B) mTOR MHC Class II (RT1 Bu) Muc-16 MHC Class II(RT1D) Muc-2 MHC Class III? 25 Muc-3 MHC Qa1b Muc-4 MICA Muc-7 MICAMICB MULT-1 MICB Munc13-4 Microfold (M) Cells Munc18 Microtubule Associated Protein 2ab 30 MUPP1 Microtubule Associated Protein RP/EB 2 Mus81 Midkine Musashi1 Mineralocorticoid Receptor Muscarinic Acetylcholine Receptor 2 MIP-12 muscle Actin MIPEP Muscleblind-like 1 Mitochondria 35 MVP Mitofilin MYBBP1A Mitofusin 1 MYBPC3 Mitofusin 2 Myc tag Mitotic Cells MyD88 MKK6 Myelin Basic Protein MLH1 Myelin oligodendrocyte glycoprotein MLK3 40 Myelin PLP MLL1 Myeloid Antigen MLLT11 Myeloid Cell Nuclear Differentiation Antigen MMP1 Myeloid Lineage MMP10 Myocilin MMP11 Myogenin MMP12 45 Myosin heavy chain MMP13 Myosin ILA MMP14 Myosin light chain 2 MMP15 Myosin light chain 3 MMP17 Myosin light chain kinase MMP19 Myosin Phosphatase MMP2 50 Myosin Phosphatase 1/2 MMP2O MYST2 MMP21 NADH2 MMP26 Naf1 MMP3 NAK MMP8 Nanog MMP9 55 NAPE-PLD Mnk1 NAT1 mNOS Native Lipoteichoic Acid MSOD Natriuretic Peptide Receptor A Moesin Natural Killer Cell Monoamine Oxidase B Natural Killer Cell Activation Structures Monocyte/Granulocyte NBS1 Mononuclear Phagocyte 60 NC1.1 Mouse Embryonic Fibroblast (mEF) Feeder Cells NCF4 Mouse Lineage Nick MPP1 NCOA1 MRCL3 NCOA2 MRE11 NCX1 MRGPR-X2 65 NDUFAF1 MRI1 NDUFB4 US 9,714,897 B2 85 86 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. NDUFS3 Nucleolin NEDD8 Nucleolin Phospho (Thr76/Thr84) NEK2 Nucleophosmin NEK6 NUDC NEK7 NUMA1 NEK9 Nurf7 NEK9 Phospho (Thr210) 10 O acetyl GD3 Nestin Oct2 NETO2 Neurabin1 Neuregulin1 Octa. Neuregulin3 ODAG Neuroblastoma 15 OGDH NeuroD1 OLIG1 NeuroD2 OLIG2 Neurofibromin Oligodendrocyte Marker Neurofilament Heavy Protein Oligodendrocyte Marker O1 Neurofilament Medium Protein Oligodendrocyte Marker O4 Neurogenin 2 Oncostatin M Receptor Neurokinin 1 Receptor Orail Neuron Specific Enolase OSCAR Neuronal Growth Factor Receptor OSR1 Neurotensin Receptor 1 Osteonectin FIB p50/p105 Osteopontin FIB pé5 (pS536) Osteoprotegerin FATc.1 25 Otx2 FIB p50 OVA (SIINFEKL) H-2Kb FIB p50/p105 Oval Cell Marker FIB p52/p100 Ovalbumin FIB p65 Ovarian Carcinoma-associated Antigen FIB pé5 (pS529) OX-62 2 30 p1101 p120 Catenin p120 Catenin (pS268) p120 Catenin (pS288) p120 Catenin (pS879) p120 Catenin (pT310) Nitrotyrosine 35 p120 Catenin (pT916) NKG2ACE p120 Catenin (pY228) NKG2AB6 13 130 NKX3.1 130 Cas NM23A p130 Cas (pY249) NMDA Receptor 2A 14ARF NMDA Receptor 2B 40 p150, 95 NMDE2 19ARF 21 NMNA2 p22phox b23 NOS p27Kip1 NNTM 45 Nociceptin Nod2 Nodal Noggin NONO p34Cdc-2 Nonspecific Cytotoxic Cells 50 38 Notch1 p38 MAPK (pT180 pY182) Notch2 3400 Notch3 S3 Notch4 p53 Acetylated (Lys305) NOX2 p53 Acetylated (Lys382) NOX4 55 p53 Phospho (Ser15) NOXA2 p53 Phospho (Ser37) NPC p53 Phospho (Ser392) NPM-ALK p53BP1 (Ser1778) NPM/B23. Phospho (Thr199) p57Kip2 NPM/B23. Phospho (Thr234/Thr237) 360 CAF1 NPYSR 62 60 63 p63 (TA) p70S6 Kinase I b90 Risk p90 Rsk Phospho (Thr368/Ser372) NSPAB 95 NBS1 NTAL 65 97 NTF97 US 9,714,897 B2 87 88 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. PABP1 5 Phospholipase C 23 PABP2 Phospholipase C I-1 PABPN1 Phospholipase D1 PAC1 Phosphoserine/threonine?tyrosine PAD2 Phosphotyrosine PAG1 PI 3 Kinase catalytic subunit It PAK1 10 PI 3 Kinase catalytic subunit 3 PAK2 PI 3 Kinase p110 I PAK3 PI 3 Kinase p110 I' pan Actin PI3 Kinase p150 pan Macrophage PI 3 Kinase p85 It Panendothelial Cell Antigen PI 4 kinase I? PAR1 15 PLAS1 Parainfluenza Virus type 1 PLAS3 Parainfluenza Virus type 2 PICK1 Parainfluenza Virus type 3 PIM1 PARC PIM2 PARD3 Pin1 PARK7.DJ1 PINK1 PARP, Cleaved Form 2O PIPSK2It PARP16 PIP5KIf PARP4 PIR-AB PARVA Pirh2 Pax2 PIST PaxS PTX3 Pax6 25 PIWIL2 Pax7 PKA RIII+ (pS99) Pax8 PKA RIII (pS114) Pax9 PKA2I2 Paxillin PKAR2 Paxillin Phospho (Tyr118) PKAf Paxillin Phospho (Tyr31) 30 PKC PBEF PKCd PBP PKCIE (pT497) PBR PKCI+ (pT638) PBX3 PKCI2 PCB 35 PKCI2 PCNA PKCI3 PD-1H PKCII PD-ECGF PKCI PDC-TREM PKCI, PDCD4 PKCI . . . PDCD6 40 PKN

PDECGF PKR.

PDI PLA2G1B PDK1 Placental alkaline phosphatase PDK2 45 Placental Protein 14 PDPK1 Plakophilin 3 PDPK1 (pS241) Plastin L PDX1 Platelet PDZK1. PLAU PE PLCl31 PECR 50 PLCI1 (py 783) PEI-Transferrinfection PLCI2 Pellino. 1 PLCI2 (py 759) Pentraxin 3 Plectin PEPD Pleiotrophin Perforin Plexin A1 Peroxiredoxin 1 55 PlexinB2 Peroxiredoxin 2 PLGF Peroxiredoxin 6 PLK1 PEX5 PLK1 Phospho (Thr210) PF4 PLK4 PGC1 it PLSCR1 PGIS PLVAP PGP9.5 60 PLZF PGRP-Ia PMCA(1-4) PGRP-S PMCA4 PHD1 PMEL17 SILV PHD2 PMN Phosphatidylserine PMP70 Phospho SHIP 65 PMS2 Phospholipase A2 activator protein (PLAP) PNA US 9,714,897 B2 89 90 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. PNPH PUMAI Podocalyxin Pumilio 1 Podoplanin Pumilio2 POKEMON Polyhistidine Tag PYCARD PON Pygopus2 10 Pyk2 (pY402) Pyruvate Dehydrogenase E1It Pyruvate Dehydrogenase E2 Pyruvate Dehydrogenase E2, E3bp q2 15 Qal(b) Qa2 B11A B2S Pre-BCR B27A Pre-T Cell Receptor I + Chain B4 Prealbumin BSa Presenilin1 B9 Presenilin2 ac1 Prion protein PrP ac1, Cdc42 PRKRA D17 PRLR D17 Phospho (Ser645) PRMT1 D23A PRMT5 25 D51 pro Relaxin 1.2 pro Relaxin 2 Profilin1 ixin Progesterone Receptor E-1 Prohibitin Prokineticin 1 30 Prokineticin 2 GE Prolactin DD ProMBP1 Rainbow Trout Ig Prostaglandin D2 Receptor RaBP1 Prostaglandin dehydrogenase 1 Ranp Prostaglandin E Receptor EP3 35 RanCAP1 Prostate Cell Surface Antigen RAP1A, RAP1B Prostate Specific Antigen RAP1GAP Prostatic Acid Phosphatase Raptor Proteasone 20S C2 RARIE Proteasome 20S I 2 RAS Proteasome 20S I + 3 RASGAP Proteasome 20S I + 5 40 RASGRF1 Proteasome 20S I + 6 RASSF1A Proteasome 20S I + 7 Rb Proteasome 20SI: 1,2,3,5,6,7 Rb (a.a. 332-344) Protein A Rb (pS780) Protein G Rb (p.S807 p.S811) Protein Kinase D2 45 RbAp46 Protein Phosphatase 112 RbAp48 Protein phosphotase inhibitor 1 RBC Protein S RBC (Polyclonal Rabbit) Proteinase Activated Receptor 4 RBM3SA Prothrombin RBP4. PSA-NCAM 50 PSD95 RCC1 Pseudomonas Aeruginosa RCRL6 PSMA Red Blood Cell PSMD14 Relaxin 1 Psoriasin Relaxin 12 PTAFR 55 Relaxin 2 PTBP1 RelB PTEN RELMI? PTGER2 RELT PTGER4 Renin PTHLH RENT1 PTK7 Reptin 60 Repulsive Guidance Molecule C Resistin PTPS REST PTPIA Ret PTRH2 Reticular Fibroblasts and Reticular Fibres PU.1 Reticulon 1A PU60 65 Reticulum Cells PUMA Retinoblastoma 1

US 9,714,897 B2 93 94 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. SMAD3 5 SULT2A1 SMAD4 SUMO1 SMADS SUMO2 SMAD6 SUMO3 SMC1 SUN1 SMC1L1 Suppressor of Fused SMN 10 SUPT16H Smoothelin Survivin SMURF2 Survivin Phospho (Thr34) SNAP2S SV40 Large T and Small t Antigens SNX1 SWC1a SOAT1 SWC6 SOCS1 15 SYBL1 SOCS2 Syk SOCS3 Syk (pY348) SOCS6 Synapsin I SOD2 Synapsin II Sodium Potassium ATPase Synaptoanin2 Sonic Hedgehog Synaptophysin Sortilin 2O Syndecan4 SOSC3 SynCAP SOX1 Synip SOX10 Syntaxin SOX17 Syntaxiné SOX18 Syntrophin SOX2 25 SYWC SOX2 (COOH terminus) T cells (pan reactive) SOX2 (NH2 terminus) T Lymphocytes SOX9 T- and B-Cell Activation Antigen SP-D T7 tag Sp1 TAB1 Sp3 30 TACE Spectrin I + 1 TACI SPHK1 TAF172 Spt16 TAF2SO Src (pY418) TAG-72 SREBP1 Talin1 SSDNA 35 Talin2 SSEA3 Tamm Horsfall (Uromucoid) SSEA4 TANK1 SSEAS TAP1 SSH3BP1 TAP2 SSR2 TARDBP SSRS TARP SSRP1 40 Tartrate-resistant acid phosphatase SSX2IP TAS1R1 Stat1 Tau Stat1 (N-Terminus) TBA1B Stat1 (pS727) Tbet Stat1 (pY701) TBK1 (pS172) Statli 45 TBX1 Stat? TC10 Stats TCF3 Stat3 (pS727) TCF7L1 Stat3 (pY705) TCF7L2 Stata TCL1 Stata (pY693) 50 TCP1i Stats TCP1i2 Stats (pY694) TCR Statsa TCR DO11.10 Statsb. TCRHY Stat6 TCR VI - 11 Stat6 (pY641) 55 TCR VI + 11.1/11.2b, d Stathmin Op18 Phospho (Ser16) TCR VI 2 Stathmin1 TCR VI + 24 Stefin B TCR VI it 24-Ji + 18 Stem Cell Factor TCRVI, 3.2 STIM1 TCR VI + 3.2b, c STK3 TCR VI + 7.2 STK33 60 TCR VI + 8 STK39 TCR VI + 8.3 STOM TCR V121 STRO1 TCR VI.10a STUB1 TCR V1210b SULT1A1 TCR Vi211 SULT1A3 SULT1A4 65 TCR V1212 SULT1C2 TCR V1212b US 9,714,897 B2 95 96 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. TCR Vi213 5 TIA-1 TCRV13.1 TIAM2 TCR VI213.2 Tie1 TCR V1213.6 Tie2 (pY1102) TCR VI-14 Tie2 (pY992) TCR VI 16 TIF1P Phospho (Ser473) TCR VI 17. 10 TIGIT TCR VI 17It Tim1 TCR VI.18 Tim2 TCR VI2 Tim3 TCRVI220 Tim3 Fc Fusion Protein TCR V121.3 Tim4 TCR VI22 15 TimSO TCR VI-23 Timeless TCRVI23 TIMP1 TCR V124 TIMP2 TCRV25 TIP49A TCR VI 5.1 TIRAP TCR VI25.1/5.2 TIS11b TCR Vi25.2 2O TL1A TCR VIP5.3 TLK1 TCR VI26 TLR11 TCR Vi27 TLR12 TCR Vi27.1 CD285 TCR V127.2 TLR7 TCRVI28 25 TLR8 TCR V8.1/8.2 TMEFF2 TCRVI28.2 TMPS2 TCR VI8.2/8.3 TMSA TCR VI's 28.4 TMTSP TCR V8.3 TNAP TCR V8.5 30 TNAP3 TCR VIP9 TNF-i- TCR VI.1.1 TNF-2 TCR Vi1.1/f 1.2 TNFR Related Protein TCR Vi2 TNPO3 TCR Vis Tollip TCR VI9 35 TOMM2O TCRVI1 TOMM22 TCRV12 TOP1 TCR VI'4 TOP2A TCRVI'6.3.2 TOP2B TCRIE TORC2 TCR i + 2 Torsin A TCR 2 40 TOX TCRii TPH1 TCR i TPPP TCTP TPTE TT TR11B Tec TRA-1-60 TEF1 45 TRA-1-60R TEM8 TRA-1-81 Tenascin C TRA-2-49 TER119 TRA-2-54 TERF2 TRADD Terminal-Deoxynucleotidyl Transferase TRAF2 TERT 50 TRAF4 Tetranectin TRAFS TFF TRAF6 TFIIB TRAM2 TGF-2 Transferrin TGF-21 Transglutaminase TG F-3 55 Transglutaminase2 TG F-PR1 Transketolase TGF-IR2 TRAP1 TGN38 TRAPPC2 TGN46 TRAPit THAP11 Trem-like 2 THEMIS Trem-like 4 Thioredoxin 60 TRIB2 Thioredoxin Reductase 1 TRIB3 TPOK TRIM Thrombin Receptor TRIM2S Thrombocyte TRIM29 Thrombospondin TRK Thymidine Kinase 1 65 TrkA Thyroglobulin TrkC

US 9,714,897 B2 101 102 TABLE 8-continued TABLE 8-continued

Cell surface markers for use with the Cell surface markers for use with the hydrogel particles described herein. hydrogel particles described herein. CEACAM4 5 WNT9a Claudin-3 5HT2C CLP24 AATK CRHR1 ACLP DC-STAMP ADAMTS15 Eph Receptor B3 alpha 1B Adrenoreceptor FATP4 10 APLP1 FcRL1 Fluorescein Oregon Green FcRL2 RXR-12

FSH-R CCL1 Gi24 PRDM4 Histamine H1 Receptor 15 ACTH NeuSGc PDZ binding kinase Lin28A HuC/HuD neuronal protein L-33 Rit TDRD3 ATM (pSer1981) EP300 integrin I 8 Carbonic Anhydrase VI integrin 27 Cholecystokinin A Receptor (integrin I28 2O CCL23

KOR Chondrollectin CD85 Chordin-Like 2 LRIG3 Claudin-10b LRP4 Claudin-11 MMP16 25 Claudin-12 MS4A4A Claudin-17 NAALADase-like 2 CLEC2A Neuropeptide Y receptor type 1 Coagulation Factor VII Oncostatin M Receptor i2 CXCL1,2,3

PEAR1 30 DPCR1 PEDF Receptor Dipeptidyl peptidase 6 Plexin A4 Epithelial membrane protein 3 Protocadherin1 Endoglycan ROBO2 Calgranulin C ROBO4 FATP2 EDG8 35 FATP5 Scavenger receptor A5 FcRLB Semaphorin 4A GLP-2R Semaphorin 4B GLUT3 Semaphorin 6A Glypicané Siglec-16 GPR-22 Somatostatin Receptor 3 GPR-37 STING 40 GPR-37L1 GPBAR1 INSRR TM4SF4 LINGO1 TMEM87A LINGO2 TSPAN2 mGluR2 VEGF-R1, 2, 3 mGluR7 ADAM15 45 MMP25 Calreticulin2 Neuromedin B Receptor Complement Factor H-related 4 NRAGE CXCL6 Osteoactivin CD158a?h/b2/fg Porimin EaS2-68 peptide bound to I-Ab Prokineticin Receptor 1 HLA-Bwa. 50 Prominin2 ATF1 Phospho (Ser63) Semaphorin 3A Epiregulin SLAP-130 FATP1 Somatostatin Receptor 5 Fibromodulin SCARF1 Furin STAMP2 Galanin 55 TAFA3 IL-11 TAFA4

MFG-E8 Tuberous Sclerosis 1 MINA TCF8 OctaA CMG2 OLIG1, 2, 3 IL-17D Receptor Oncostatin M 60 Macrophage Stimulating Protein Receptor Semaphorin 3E Siglec-11 Slug Syndecan3

STYK1 CD8Se. LTBP1 SOX7 TIMP3 65 Activin A Receptor Type IA WAP-B Carbohydrate Sulfotransferase 15

US 9,714,897 B2 105 106 TABLE 8-continued examples, a target cell can be a primary neuron; established neuron; transformed neuron; stably transfected neuron; or Cell surface markers for use with the hydrogel particles described herein. motor or sensory neuron. In other embodiments, a target cell is selected from the GILT Recoverin group consisting of primary lymphocytes, monocytes, and Cardiac Troponin I granulocytes. A target cell can be virtually any type of cell, including HLA-B7 B27 prokaryotic and eukaryotic cells. Myosin light chain 2a 10 Myosin light chain 2V Suitable prokaryotic target cells include, but are not Epithelial Antigen limited to, bacteria Such as E. coli, various Bacillus species, CD791 + cy and the extremophile bacteria Such as thermophiles. CD92 Suitable eukaryotic target cells include, but are not limited 15 to, fungi such as yeast and filamentous fungi, including In one embodiment, a plurality of hydrogel particles is species of Saccharomyces, Aspergillus, Trichoderma, and used to determine the dynamic range and/or sensitivity of Neurospora; plant cells including those of corn, Sorghum, detection of a particular cell Surface marker or combination tobacco, canola, soybean, cotton, tomato, potato, alfalfa, thereof on a population of target cells. For example, the Sunflower, etc.; and animal cells, including fish, birds and population of hydrogel particles can be tuned to have the mammals. Suitable fish cells include, but are not limited to, SSC and/or FSC profile of the target cell, and subpopula those from species of Salmon, trout, tilapia, tuna, carp, tions of the hydrogel particle are derivatized with a specific flounder, halibut, Swordfish, cod and Zebrafish. Suitable bird number of copies of a cell Surface marker, e.g., a cell Surface cells include, but are not limited to, those of chickens, ducks, receptor, or a domain thereof, for example, an epitope quail, pheasants and turkeys, and other jungle foul or game binding region thereof. For example, individual Subpopula 25 birds. Suitable mammalian cells include, but are not limited tions of hydrogel particles can each be derivatized to have a to, cells from horses, cows, buffalo, deer, sheep, rabbits, unique number of copies, e.g., one Subpopulation will con rodents such as mice, rats, hamsters and guinea pigs, goats, tain 100 copies of a cell Surface marker, a second subpopu pigs, primates, marine mammals including dolphins and lation will contain 1,000 copies of the same cell surface marker, a third subpopulation will contain 10,000 copies of 30 whales, as well as cell lines, such as human cell lines of any the same cell Surface marker, etc. The populations of hydro tissue or stem cell type, and stem cells, including pluripotent gel particles are fluorescently stained for the respective cell and non-pluripotent, and non-human Zygotes. Surface marker and fluorescence is detected for hydrogel Suitable cells also include those cell types implicated in a particles in each subpopulation. In this regard, the Subpopu wide variety of disease conditions, even while in a non lations of hydrogel particles can be used to generate a 35 diseased State. Accordingly, Suitable eukaryotic cell types standard curve of fluorescence emission for target cells with include, but are not limited to, tumor cells of all types (e.g., the respective cell marker. The cell surface marker can be melanoma, myeloid leukemia, carcinomas of the lung, any of the cell surface markers provided thereof, or binding breast, ovaries, colon, kidney, prostate, pancreas and testes), regions thereof, or a cell Surface marker known to one of cardiomyocytes, dendritic cells, endothelial cells, epithelial ordinary skill in the art. 40 cells, lymphocytes (T-cell and B cell), mast cells, eosino Hydrogel particles of the disclosure behave similarly to phils, vascular intimal cells, macrophages, natural killer target cells in procedures such as staining and analysis by cells, erythrocytes, hepatocytes, leukocytes including mono flow cytometry or FACS. For example, in one embodiment, nuclear leukocytes, stem cells such as hematopoietic, neural, a hydrogel particle has one or more optical properties skin, lung, kidney, liver and myocyte stem cells (for use in substantially similar to one of the cell types set forth in Table 45 screening for differentiation and de-differentiation factors), 1, Table 2 or Table 3. osteoclasts, chondrocytes and other connective tissue cells, In some embodiments, a target cell is an immune cell. keratinocytes, melanocytes, liver cells, kidney cells, and Non-limiting examples of immune cells include B lympho adipocytes. In certain embodiments, the cells are primary cytes, also called B cells, T lymphocytes, also called T cells, disease state cells, such as primary tumor cells. Suitable natural killer (NK) cells, lymphokine-activated killer (LAK) 50 cells also include known research cells, including, but not cells, monocytes, macrophages, neutrophils, granulocytes, limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See mast cells, platelets, Langerhans cells, stem cells, dendritic the ATCC cell line catalog, hereby expressly incorporated by cells, peripheral blood mononuclear cells, tumor infiltrating reference. (TIL) cells, gene modified immune cells including hybrido mas, drug modified immune cells, and derivatives, precur 55 In some embodiments, a target cell is a tumor microve sors or progenitors of any of the cell types listed herein. sicle or tumor macrovesicle. Tumor microvesicles, also In some embodiments, a target cell encompasses all cells known as tumor-secreted microvesicles or tumor-Secreted of a particular class of cell with shared properties. For exosomes, can be found in circulating blood and may have example, a target cell can be a lymphocyte, including NK immune-suppressive activities. Tumor microvesicles typi cells, T cells, and B cells. A target cell can be an activated 60 cally range in size from 30-200 nm in diameter. Larger lymphocyte. tumor micro vesicles may be referred to as tumor macro In some embodiments, a target cell is a primary cell, vesicles, and can range in size from 3-10 um in diameter. cultured cell, established cell, normal cell, transformed cell, The hydrogel particles described herein can be employed infected cell, stably transfected cell, transiently transfected in any flow cytometer known to those of ordinary skill in the cell, proliferating cell, or terminally differentiated cells. 65 art. For example, one or more of the flow cytometers In one embodiment, a target cell is a primary neuronal provided in Table 9 below are amenable for use with the cell. A variety of neurons can be target cells. As non-limiting hydrogels and assays described herein. US 9,714,897 B2 107 108 TABLE 9 TABLE 9-continued

Instruments for use with embodiments described herein Instruments for use with embodiments described herein nStrument Manufacturer Instrument Manufacturer MACSQuant (R) Analyzer 10 Miltenyi CyFlow (R) SL Partec technology MACSQuant (R) VYB Miltenyi CyFlow (R) Sorter Partec technology BD FACSCalibur TM BD Biosciences CyFlow (R) CCA Partec technology BD FACSCanto TM High Throughput Sampler BD Biosciences CyFlow (R. Oenolyser Partec technology BD FACSCanto II BD Biosciences NucleoCounter (R) NC-3000TM Chemometec BD FACSCanto TM BD Biosciences 10 NucleoCounter (R) NC-250 TM Chemometec BD FACSCount TM BD Biosciences NucleoCounter (R) NC-200 TM - High Precision Cell Chemometec BD AccriTM C6 BD Biosciences Counter BDLSRFortessa TMX-20 BD Biosciences HPC-100 Portable Flow Cytometer Cronus BD FACSCanto TM II BD Biosciences Technologies Ltd BD LSR II BD Biosciences Cytell Cell Imaging System GE Healthcare BDLSRFortessa TM BD Biosciences 15 MAGPIX Luminex BD FACSVerse TM BD Biosciences Luminex (R) 100/200 TM System Luminex BD FACSAria TM Fusion BD Biosciences FLEXMAP 3D (R) Luminex BD FACSAria TM BD Biosciences ImageXpress (R) Velos Laser Scanning Cytometer molecular devices BD FACSAria TM II BD Biosciences ClonePix TM 2 molecular devices BD FACSJazz TM BD Biosciences SpectraMax (R) i3 molecular devices BD InfluxTM BD Biosciences AQ1 Discrete Analyzer SEAL Analytica Fortessa XSO. BD Biosciences FlowSight Flow Cytometer Millipore AQ2 Discrete Analyzer s EAL Analytica Guava easyCyte 6-2L Benchtop Flow Cytometer Millipore guava easyCyte 5HT Benchtop Flow Cytometer Millipore AQ400 Discrete Analyzer s EA L. Analytica guava easyCyte 8 Benchtop Flow Cytometer Millipore guava easyCyte 5 Benchtop Flow Cytometer Millipore AQUA 900 s EA L. Analytica guava easyCyte 8HT Benchtop Flow Cytometer Millipore 25 guava easyCyte 6HT-2L Benchtop Flow Millipore AA3 HR AutoAnalyzer s EA L. Analytica Cytometer ImageStreamX Mark II Imaging Flow Cytometer Millipore AA1 AutoAnalyzer s EA L. Analytica Muse Cell Analyzer Millipore guava easyCyte 12HT Benchtop Flow Cytometer Millipore QuAAtro39 s EA L. Analytica guava easyCyte 12 Benchtop Flow Cytometer Millipore 30 S3e TM Ce Sorter Bio-Rad Infralyzer 2000 s EA L. Analytica S3 TM Ce Sorter Bio-Rad Avalon Cell Sorter Bio-RadPropel Technicon AutoAnalyzer II (AAII) s EA L. Analytica Labs CytoFLEX Beckman Coulter Technicon Bran + Luebbe TrAAcs 800-2000 s EAL Analytica FP 1000 Cell Preparation System Beckman Coulter 35 Vi-CELL (R) XR Cell Viability Analyzer Beckman Coulter Bran + Luebbe FIA Analyzer SEAL Analytica FC 500 Series Beckman Coulter MoFlo (R) Astrios TM Beckman Coulter BioSorter (R) Large Particle Flow Cytometer Union Biometrica, Coulter Epics XLTM and XL-MCLTM Beckman Coulter IlC. Gaios TM Beckman Coulter COPASTM Large Particle Flow Cytometers Union Biometrica, CyAn TM ADP Analyzer Beckman Coulter IlC. Attune TM Acoustic Focusing Cytometer Life Technologies 40 Cellometer Mini Cell Counter Nexcelom Attune (R) NXT Acoustic Focusing Cytometer Life Technologies Cellometer Auto T4 Cell Viability Counter Nexcelom EVOS Life Technologies Cellometer Auto X4 Cell Viability Counter Nexcelom Countess IIFL Life Technologies Cellometer Auto 1000 Cell Viability Counter Nexcelom EC800 Cell Analyzer Sony Cellometer Auto 2000 Cell Viability Counter Nexcelom SH800 Cell Sorter Sony Cellometer Vision CBA Nexcelom SP6800 Spectral Analyzer Sony 45 Celigo S Nexcelom SY32OO Ce Sorter Sony NovoCyte TM 1000 ACEA A50-Micro Apogee Flow NovoCyte TM 2000 ACEA Systems NovoCyte TM 2060 ACEA A50-Universal Apogee Flow NovoCyte TM 3000 ACEA Systems HPC-100 Handyem Auto40 Apogee Flow 50 S1 OOOEX Stratedigm Systems SES2OX Stratedigm FlowSight Amnis Sysmex (R DI-60 Sysmex ImageStream Mark II Amnis Cella Vision (RDM96 Sysmex JSAN Bay Bioscience Cella Vision (RDM1200 Sysmex CytoSense CytoBuoy Cytation BioTek CytoSub CytoBuoy 55 EasyCell Assistant Medica CytoSense CytoBuoy IN Cell Analyzer GE Healthcare CytoBuoy CytoBuoy Fluorish List Cytonome Viva TM G1 CYTONOME GigaSort TM CYTONOME Big Blue BD Biosciences Hydris CYTONOME Kermit Miltenyi Agilent 2100 Bioanalyzer Agilent ac6 BD Biosciences Technologies 60 SrDAS BD Biosciences NovoCyte ACEA Biosciences 8. BD Biosciences CyFlow (R) Space Partec technology FACSCanto II Immunology BD Biosciences CyFlow (R) Cube 8 Partec technology Test Cyt Millipore CyFlow (R) Cube 6 Partec technology milt Miltenyi CyFlow (R) Ploidy Analyser Partec technology 80 BD Biosciences CyFlow (R) Counter Partec technology 65 ietest BD Biosciences CyFlow (R) miniPOC Partec technology Curiel's Aria BD Biosciences US 9,714,897 B2 109 110 TABLE 9-continued TABLE 9-continued

Instruments for use with embodiments described herein Instruments for use with embodiments described herein

nStrument Manufacturer nStrument Manufacturer AttuneA (R Acoustic Focusing Cytometer Life Technologies 4 Laser LSR II BD Biosciences Blue Violet 5 Laser LSR II BD Biosciences Medawar LSRII BD Biosciences FACSArray BL-2 BD Biosciences Medawar Calibur BD Biosciences FACSCalibur BD Biosciences FACSAria INER BD Biosciences DUAL for long term studies BD Biosciences Attune RA Life Technologies 10 MoFlo 1095 Production only Beckman Coulter Fortessa BD Biosciences BL-2 FACSAria III Sorter BD Biosciences Aria BD Biosciences Astrios BL-2 sorter Beckman Coulter SORTER BD Biosciences Tessy BD Biosciences Cyan Beckman Coulter LSR. II-1 BD Biosciences LSR II BD Biosciences Fortessa BD Biosciences ARIA BD Biosciences 15 4 laser AriaIII BD Biosciences Canto II BD Biosciences LSRFortessa BD Biosciences FO9 - LSR. Fortessa. 1 BD Biosciences UoN FACSAria II cell sorter BD Biosciences The Hof BD Biosciences Door Beckman Coulter 6th Floor Hess Fortessa A BD Biosciences Fortessa BD Biosciences Cerebro BDFACSAriaII BD Biosciences WCI - FACSAria I BD Biosciences Mystique BDFACSArial II BD Biosciences LSRII Karp8 BD Biosciences Godzilla BDFACSAriaII BD Biosciences Karp 8 BD Biosciences Wolverine BDFACSAriaII BD Biosciences Canto BD Biosciences Megatron BDFACSAriaII BD Biosciences Aria sorter BD Biosciences Megatron BDFACSAriaII BD Biosciences DI lab BD Biosciences Fortessa B BD Biosciences DIFACSAria BD Biosciences 6 colour Canto II BD Biosciences Constance BD Biosciences O colour LSR II BD Biosciences 25 DIFACSAria III BD Biosciences 4 laser 13 colour Influx sorter BD Biosciences WCI FACS Canto BD Biosciences 4 colour X20 BD Biosciences MACSQuant 10 Miltenyi SORP BD Biosciences VAMC Memphis LSR BD Biosciences FACSAria INER BD Biosciences VAMC Memphis S3 Bio-Rad LSRS61 BD Biosciences ARIA INER BD Biosciences Fortessa FCF UZH BD Biosciences 30 Uhura BD Biosciences LSR 2 B BD Biosciences Kirk BD Biosciences LSRII-C BD Biosciences Data Millipore Cal 3 BD Biosciences Spock BD Biosciences Aria II A BD Biosciences McCoy BD Biosciences LSR 16 BD Biosciences LSB Fortessa BD Biosciences 35 MMUNLSRI BD Biosciences RC BD Biosciences EXAMPLES UVLSR BD Biosciences 5 Laser Aria BD Biosciences Curiel's LSR II BD Biosciences The present invention is further illustrated by reference to LSR. Fortessa BD Biosciences the following Examples. However, it should be noted that Mauzeroll Aria BD Biosciences 40 Frenette BD Biosciences these Examples, like the embodiments described above, are Fallon Beckman Coulter illustrative and are not to be construed as restricting the Galios Beckman Coulter Scope of the invention in any way. LSRIIFortessa BD Biosciences FACSCanto II CLSB BD Biosciences LSR II SC BD Biosciences 45 Example 1: Generation of Hydrogel Particles UNCAFortessa BD Biosciences VERSE BD Biosciences Photomasks for UV lithography were sourced from ARIAII BD Biosciences ARIAIII BD Biosciences CADart Services Inc. and were designed using AutoCad FO9 - BD LSRFortessa BD Biosciences (AutoDesk, Inc.). SU-8 photo resist (Microchem, Inc.) was HMRIFACSCanto IIA BD Biosciences 50 photo crosslinked on 4" silicon wafers using a collimated HMRI FACSCantoII B (HTS) BD Biosciences HMRI Aria III BD Biosciences UV light source (OAI. Inc.) to create masters for microflu L2 BD Biosciences idic device fabrication. PDMS (polydimethylsiloxane, UoN Canto BD Biosciences Sigma Aldrich, Inc.) was prepared and formed using stan LSRII M902 BD Biosciences dard published methods for soft lithography and microflu Fortessa. 1 BD Biosciences 55 F05 - FACSAria BD Biosciences idic device fabrication (See, McDonald J C, et al., 2000, FO2 - FACSAria III BD Biosciences Electrophoresis 21:27-40). F10 - BDFACSAria III BD Biosciences Droplets were formed using flow-focusing geometry FO3 - Guava Millipore where two oil channels focus a central stream of aqueous Aria Blue 11 Color BD Biosciences Aria Red BD Biosciences monomer solution to break off droplets in a water-in-oil Aria Orange BD Biosciences 60 emulsion. A fluorocarbon-oil (Novec 7500 3M, Inc.) was Aria Cyan BD Biosciences used as the outer, continuous phase liquid for droplet for Aria Emerald BD Biosciences mation. To stabilize droplets before polymerization, a sur Aria Silver BSL3 BD Biosciences LSR. Fortessa BD Biosciences factant was added at 0.5% w/w to the oil phase (ammonium LSR II Bldg 4 BD Biosciences carboxylate salt of Krytox 157 FSH, Dupont). To make the LSR Fortessa blog 4 BD Biosciences 65 basic polyacrylamide gel particle, a central phase of an CANTO II Bldg 50 BD Biosciences aqueous monomer Solution containing N-acrylamide (1-20% w/v), a cross-linker (N.N'-bisacrylamide, 0.05-1% US 9,714,897 B2 111 112 w/v), an accelerator, and ammonium persulfate (1% w/v) added to a polymer mix containing 20%. 19:1 (acrylamide: was used. An accelerator. (N.N.N',N'tetramethylethylenedi bis-acrylamide) and 0.1% allylamine in water. 0.4% ammo amine (2% vol%) was added to the oil-phase in order to niumpersulfate was added to the mix prior to droplet for trigger hydrogel particle polymerization after droplet for mation. Hydrogel particles were formed as described in mation. Example 1. Hydrogel particles with 200 g/mL of encapsu Several co-monomers were added to the basic gel formu lated calf thymus DNA displayed cell-like staining using lation to add functionality. Allyl-amine provided primary propidium iodide as visualized using a commercial imaging amine groups for secondary labeling after gel formation. We cytometer and compared to Chinese Hamster Ovary cells modulated forward scatter by adjusting the refractive index stained using the same procedure. Images were obtained of the gel by adding co-monomers allyl acrylate and allyl 10 using a Nexcelom CellometerTM (FIG. 6). methacrylate. Side scattering of the droplets was tuned by Cells obtained from a buccal Swab were washed in PBS adding a colloidal suspension of silica nanoparticles and/or and stained with propidium iodide. In parallel, populations PMMA (poly(methyl methacrylate)) particles (~100 nm) to of hydrogel particles containing a range of DNA concentra the central aqueous phase prior to polymerization. tions were also stained in the same manner. Both the cell and Stoichiometric multiplexing of the hydrogel particles was 15 particle Suspensions were analyzed on a flow cytometer achieved by utilizing co-monomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epox (488/590 nm excitation/emission). Flow cytometry analysis ide, alkyne, etc.) for secondary labeling. of cheek cells and the same range of encapsulated DNA Droplets were formed at an average rate of 5 kHz and particles showed that the particles display a range of cell were collected in the fluorocarbon oil phase. Polymerization like fluorescent properties (FIG. 7, left panel). The intensity was completed at 50° C. for 30 minutes, and the resulting of staining shows a linear correlation with the median hydrogel particles were washed from the oil into an aqueous intensity as measured by flow cytometry (FIG. 7, right Solution. panel). Example 2: Generation and Visualization of 12 11 25 Example 6: Tuning of Hydrogel Particle Side m Hydrogel Particles Scattering Water containing 5% acrylamide, 0.25% bisacrylamide, Colloidal silica was added at 12.5%, 6.25%, 3.125% and 0.05% allyl amine, and 0.1% ammonium persulfate was 0% to the aqueous fraction of the polymer mix and hydrogel flowed through a center channel and focused by oil contain 30 particles were formed as described in Example 1. Forward ing 0.1% TEMED through a 10 micron nozzle to produce 10 and side scattering data were obtained using a flow cytom um hydrogel particles, shown in FIG. 3A. Following polym eter. The results showed that side scatter signal (FIG. 8, left erization, the particles were washed in water, shown in FIG. panel) increased with higher percentages of encapsulated 3B, and conjugated to dyes of interest. The fluorescent nanoparticles while forward scatter (FIG. 8, right panel) hydrogel particles were visualized with fluorescence micros 35 remained generally unchanged, demonstrating the indepen copy, shown in FIG. 3C. dent tuning of side scatter and forward Scatter. Example 3: Multidimensional Tuning of Hydrogel Example 7: Tuning of Hydrogel Particle Forward Particle Optical Properties Scattering 40 As depicted in FIG. 4, hydrogel particles are tuned in In this experiment, the percentage of acrylamide:bis multiple dimensions to match specific cell types unlike acrylamide in the hydrogel composition was varied from polystyrene beads. Cells are deconvolved using combina between 10 and 40% to tune the refractive index of the tions of optical parameters such as FSC and SSC (FIG. 4A) hydrogel particles as measured by forward Scattering in a or secondary markers. Hydrogel particles are tuned to match 45 flow cytometer. As shown in FIG. 9, the forward scattering the SSC and FSC of specific cell types unlike polystyrene increased with increasing percentages of acrylamide: bi beads (brown) which are limited in size (FSC) and side sacrylamide as a fraction of water. scattering (FIG. 4B). Hydrogel particles are further func tionalized with stoichiometrically tuned ratios of specific Example 8: Tuning of Hydrogel Particle Optical chemical side-groups and secondary labels allowing the cell 50 Properties type to be precisely matched without suffering from bio logical noise as fixed cell lines do (FIG. 4C). An example of tuning hydrogel particles to match optical properties of a desired cell Subtype. Cof monomers can be Example 4: Flow Cytometer Delay Time as a combined with nanoparticles to tune both forward and side Function of Hydrogel Particle Diameter 55 scatter properties of the hydrogels using passive optical measurements in a flow cytometer. By combining these As shown in FIG. 5, the inter-drop delay for a flow properties with chemically labile co-monomers (e.g. allyl cytometer can be precisely correlated to hydrogel particle amine, acrylic acid), additional fluorophores/proteins/bio diameter. Data are shown for hydrogel particles of 3, 6, 10. logical side groups can be added and labeled (if desired) in 32, and 50 um diameters using flow cytometer nozzle sizes 60 order to match cell Subpopulation staining in addition to of 70 and 100 um. scattering properties. These are the three primary metric by which cells are identified using flow cytometry. Additional Example 5: Comparison of Hydrogel Particles with side groups, such as those containing heavy metals, can be Encapsulated DNA to Cells used for Cy-TOF (cytometry, time of flight mass spectrom 65 etry) calibration for example. Finally, biocompatible mate To form hydrogel particles with encapsulated DNA, 40 rial can be encapsulated to mimic Subcellular organelle ug/mL-1000) ug/mL of reconstituted calf thymus DNA was staining. US 9,714,897 B2 113 114 Example 9: Tuning of Hydrogel Particle Optical prising a polymerized monomer and being Properties functionalized such that it includes at least one cell Surface marker, A 50 nm nanoparticle colloidal Suspension was incorpo measuring the optical-scatter and hydrodynamic proper rated into the hydrogel matrix to mimic the optical proper ties of the hydrogel particle using the cytometric device ties of lymphocytes and monocytes (FIGS. 13A and 13B). to calibrate the cytometric device: The percent composition of the Suspension was altered to inserting a sample in the cytometric device, the sample match the blood cell subpopulations from the blood sample comprising a plurality of cells; control (Streck) (FIG. 13C). measuring the optical-scatter and hydrodynamic proper 10 ties of individual cells of the plurality of cells; and Specifically, the concentration of the acrylamide mono determining, based on the measurements of the optical mer (0.7-0.8M) of the hydrogel particle was adjusted to Scatter and hydrodynamic properties, whether a white increase the forward scatter of the particles to match blood blood cell or plurality thereof is present in the sample. cell Subpopulations. The percentage of bisacrylamide cross 3. The method of claim 2, further comprising sorting the linker can also be changed to affect forward scatter (1-5%). 15 white blood cell or plurality of white blood cells to a Silica nanoparticles were used at 5% or 10% in the compo separate vessel if one or more white blood cells is deter sitions to adjust side scatter. The results of this experiment mined to be present in the sample. are shown in FIG. 13. 4. The method of claim 2, further comprising: inserting a All, documents, patents, patent applications, publications, plurality of hydrogel particles into the cytometric device, product descriptions, and protocols which are cited through wherein each hydrogel particle of the plurality of hydrogel out this application are incorporated herein by reference in particles has at least one optical-scatter or hydrodynamic their entireties for all purposes. property substantially similar to a white blood cell sub-type, The embodiments illustrated and discussed in this speci wherein the cytometric device is calibrated based on mea fication are intended only to teach those skilled in the art the Sured optical-scatter and hydrodynamic properties of each best way known to the inventors to make and use the 25 hydrogel particle of the plurality of hydrogel particles. invention. Modifications and variation of the above-de 5. The method of claim 4, wherein at least one optical scribed embodiments of the invention are possible without scatter property is the same for each hydrogel particle of the departing from the invention, as appreciated by those skilled plurality of hydrogel particles. in the art in light of the above teachings. It is therefore 6. The method of claim 2, wherein at least one optical understood that, within the scope of the claims and their 30 scatter property is side scatter (SSC). equivalents, the invention may be practiced otherwise than 7. The method of claim 2, wherein at least one optical as specifically described. scatter property is forward scatter (FSC). 8. The method of claim 2, wherein the hydrogel particle The invention claimed is: has a fluorescence emission Substantially similar to a white 1. A method for determining if a sample includes one or 35 blood cell, the method further comprising: more of lymphocytes and monocytes, comprising: measuring the fluorescence emission of the hydrogel inserting, into a cytometric device, a plurality of hydrogel particle using the cytometric device to calibrate the particles Suspended in an aqueous solution, the plural cytometric device; and ity of hydrogel particles including: (1) a first portion of measuring the fluorescence emission of individual cells of hydrogel particles having forward Scatter, side scatter, 40 the plurality of cells, wherein determining whether a and hydrodynamic properties Substantially similar to a white blood cell or plurality thereof is present in the lymphocyte, and (2) a second portion of hydrogel sample is further based on measured fluorescence emis particles having forward scatter, side scatter, and sions. hydrodynamic properties substantially similar to a 9. The method of claim 2, wherein at least one optical monocyte, wherein each hydrogel particle of the plu 45 scatter property is a ratio of FSC to SSC. rality of hydrogel particles comprises a polymerized 10. The method of claim 2, wherein the polymerized monomer and has at least one functionalized surface monomer of the hydrogel particle is selected from hydroxy having at least one cell Surface marker, ethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl measuring the forward Scatter, side scatter, and hydrody methacrylate (HEMA), propylene glycol methacrylate, acry namic properties of each hydrogel particle of the plu 50 lamide, N-vinylpyrrolidone (NVP), methyl methacrylate, rality of hydrogel particles using the cytometric device; glycidyl methacrylate, glycerol methacrylate (GMA), glycol calibrating the cytometric device based on the measured methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl forward Scatter, side scatter, and hydrodynamic prop methacrylate, hydroxyethoxyethyl methacrylate, hydroxydi erties of the plurality of hydrogel particles; ethoxyethyl methacrylate, methoxyethyl methacrylate, adding a sample comprising a plurality of cells to the 55 methoxyethoxyethyl methacrylate, methoxydiethoxyethyl calibrated cytometric device; and methacrylate, poly(ethylene glycol) methacrylate, methoxy measuring the forward Scatter, side scatter, and hydrody poly(ethylene glycol) methacrylate, methacrylic acid, namic properties of each cell of the plurality of cells Sodium methacrylate, glycerol methacrylate, hydroxypropyl using the calibrated cytometric device to determine if methacrylate, hydroxybutyl methacrylate, phenyl acrylate, the sample includes one or more of lymphocytes and 60 phenyl methacrylate, benzyl acrylate, benzyl methacrylate, monocytes. 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phe 2. A method for detecting a white blood cell in a sample, noxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylth comprising: ioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribro inserting, into a cytometric device, a hydrogel particle in mophenyl acrylate, 2,4,6-tribromophenyl methacrylate, aqueous solution, the hydrogel particle having optical 65 pentabromophenyl acrylate, pentabromophenyl methacry Scatter and hydrodynamic properties Substantially simi late, pentachlorophenyl acrylate, pentachlorophenyl meth lar to a white blood cell, the hydrogel particle com acrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl US 9,714,897 B2 115 116 methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, sinistrin, sizofiran, welan gum, xanthan gum, Xylan, xylog 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, lucan, Zymosan, or a combination thereof. 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 16. The method of claim 11, wherein the biodegradable 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl monomer is chitosan or hyaluronan. methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxy 17. The method of claim 12, wherein the protein is a ethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl Structural protein, a domain thereof, or a combination methacrylamide, N-benzyl acrylamide, N-benzyl methacry thereof. lamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacry 18. The method of claim 12, wherein the protein is a lamide, N-diphenylmethyl acrylamide N-(4-methylphenyl) proteoglycan, a domain thereof, or a combination thereof. methyl acrylamide, N-1-naphthyl acrylamide, N-4- 10 19. The method of claim 12, wherein the protein is an nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, extracellular matrix component. N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acryl 20. The method of claim 18, wherein the proteoglycan is amide, N.N-methylphenyl acrylamide, N.N-phenyl phenyl decorin, biglycan, testican, bikunin, fibromodulin, lumican, ethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4- a domain thereof, or a combination thereof. methyl phenyl)methyl methacrylamide, N-1-naphthyl 15 21. The method of claim 19, wherein the protein is elastin methacrylamide, N-4-nitrophenyl methacrylamide, N-(2- or a proteoglycan. phenylethyl)methacrylamide, N-triphenylmethyl methacry 22. The method of claim 19, wherein the protein is lamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methyl collagen. phenyl methacrylamide, N,N'-phenyl phenylethyl 23. The method of claim 22, wherein the collagen is methacrylamide, N-vinyl carbazole, 4-vinylpyridine, 2-vi collagen type I, collagen type II, collagen type III, a domain nylpyridine, or a combination thereof. thereof or a combination thereof. 11. The method of claim 2, wherein the polymerized 24. The method of claim 2, wherein the polymerized monomer of the hydrogel particle is a biodegradable mono monomer of the hydrogel particle is bifunctional. le. 25. The method of claim 2, wherein the at least one cell 12. The method of claim 11, wherein the biodegradable 2 surface marker is one of the cell surface markers set forth in monomer is a monosaccharide, disaccharide, polysaccha Table 4, or a combination thereof. ride, peptide, protein, or protein domain. 26. A method for calibrating a cytometric device for 13. The method of claim 12, wherein the biodegradable analysis of white blood cells, comprising: monomer is a protein or protein domain comprising at least inserting, into a cytometric device, a plurality of hydrogel one non-natural amino acid. 30 particles suspended in an aqueous solution, each hydro 14. The method of claim 11, wherein the biodegradable gel particle of the plurality of hydrogel particles having monomer is a structural polysaccharide. side scatter, forward scatter, and hydrodynamic prop 15. The method of claim 11, wherein the biodegradable erties substantially similar to one of a lymphocyte, monomer is agar, agarose, alginic acid, alguronic acid, alpha monocyte, or granulocyte, each hydrogel particle com glucan, amylopectin, amylose, arabinoxylan, beta-glucan, 35 prising a polymerized monomer and configured with at callose, capsullan, carrageenan polysaccharide, cellodextrin, least one functionalized surface having at least one cell cellulin, cellulose, chitin, chitosan, chrysolaminarin, curd surface marker; lan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, measuring the side scatter, forward scatter, and hydrody ficoll, fructan, fucoidan, galactoglucomannan, galactoman namic properties of each hydrogel particle of the plu nan, galactosaminoogalactan, gellan gum, glucan, gluco 40 rality of hydrogel particles using the cytometric device: mannan, glucorunoXylan, glycocalyx, glycogen, hemicellu and lose, homopolysaccharide, hypromellose, icodextrin, inulin, calibrating the cytometric device for analysis of white kefiran, laminarin, lentinan, levan polysaccharide, lichenin, blood cells based on the measured side scatter, forward mannan, mixed-linkage gluxan, paramylon, pectic acid, pec scatter, and hydrodynamic properties of the plurality of tin, pentastarch, phytoglycogen, pleuran, polydextrose, 45 hydrogel particles. polysaccharide peptide, porphyran, pullulan, schizophyllan,