A Haptotaxis Assay for Leukocytes Based on Surface-Bound Chemokine Gradients Ina Rink, Jan Rink, Dorothea Helmer, Daniel Sachs and Katja Schmitz This information is current as of September 28, 2021. J Immunol published online 27 April 2015 http://www.jimmunol.org/content/early/2015/04/25/jimmun ol.1500148 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2015, doi:10.4049/jimmunol.1500148 The Journal of Immunology

A Haptotaxis Assay for Leukocytes Based on Surface-Bound Chemokine Gradients

Ina Rink,* Jan Rink,† Dorothea Helmer,‡ Daniel Sachs,* and Katja Schmitz*

The migration of leukocytes in response to chemokine gradients is an important process in the homeostasis of the human and inflammation. In vivo the migration takes place on the surface of the endothelium to which the chemokine gradient is immobilized via interaction with glycosaminoglycans. To study leukocyte migration in response to surface-bound chemokines, we generated chemokine gradients by a simple stamping method: agarose stamps were soaked with chemokine solution to form con- tinuous chemokine gradients by diffusion. These gradients could be easily transferred to a petri dish surface by stamping. We show that neutrophil granulocytes recognize these gradients and migrate toward increasing chemokine concentrations dependent on the slope of the gradient. Single-cell responses were recorded, and statistical analyses of cell behavior and migration were performed.

For analysis of /haptotaxis, we propose a chemotactic precision index that is broadly applicable, valid, and allows for a Downloaded from straightforward and rapid quantification of the precision by which cells follow the direction of a given gradient. The presented tech- nique is very simple, cost-efficient, and can be broadly applied for generating defined and reproducible immobilized gradients of almost any protein on surfaces, and it is a valuable tool to study haptotaxis. The Journal of Immunology, 2015, 194: 000–000.

hemokines are small, secreted signal proteins that play Zigmond chamber (21), the Dunn chamber (22), and the under- a key role in the human immune response, as well as agarose assay (23) have been established for this purpose. How- http://www.jimmunol.org/ C inflammatory and autoimmune diseases and development ever, most of these assays are liable to limitations such as a lack of (1–4). A well-known and thoroughly studied representative is the control due to temporal instability of the gradient or the ob- CXCL8 (IL-8). Since its first description in 1987 it has been served cell behavior. Another approach for generating gradients is known as a neutrophil-activating protein that leads to two in vivo the use of microfluidic devices that enable the composition of effects on neutrophils, chemotaxis and release of granule enzymes controllable, spatially and temporally defined concentration gra- (5, 6). CXCL8 mediates its chemotactic effect on neutrophils by dients (24–27). Therefore, it is applied for chemotaxis experi- binding its two cognate G protein–coupled receptors, CXCR1 and ments and allows the study at single-cell levels and in real time CXCR2 (7–9). Endothelial cells present glycosaminoglycans (28–30). These assays do not fully reflect the in vivo situation

(GAG) on their cell surface to which CXCL8 is able to bind via its where the migration of neutrophils takes place on the endothelium by guest on September 28, 2021 C-terminal GAG binding site (10, 11). Due to the GAG interac- triggered by the activation of surface-bound chemokines (31). An tion, a stable CXCL8 gradient is formed on the surface of the assay for migration on a surface poses a basis for research of the endothelial cells and activated neutrophils migrate along this response of neutrophils to immobilized chemokines and would gradient toward the site of inflammation (12, 13). Its critical role complement the existing chemotaxis assays. Microcontact printing in acute inflammation (14) makes it an interesting and important has been widely used to immobilize protein gradients and patterns target for therapy due to the involvement of CXCL8 in many on surfaces (32–34) and to determine the influence of immobilized chronic inflammatory diseases like rheumatism (15, 16), chronic proteins on cell adhesion, growth, and migration (35, 36). It was bronchitis (17), multiple sclerosis (18), and Crohn’s disease (19). shown that the biological activity of the chemokine can be pre- An increased understanding of the leukocyte response to immo- served upon immobilization on a surface (37, 38). Microfluidic bilized chemokines will contribute to the development of new networks can also be used for patterning protein gradients onto diagnostic tools and therapeutic approaches. surfaces (39–41). The use of hydrogels for microcontact printing Chemotaxis, that is, cell migration in response to gradients of instead of polydimethylsiloxane has been previously reported (42, soluble chemoattractants, is a commonly studied cell response. 43). Mayer et al. (44) have shown that gradients of BSA formed Several migration assays like the Boyden chamber (20), the in hydrogels by diffusion could be transferred onto surfaces by stamping. In this study, we demonstrate that continuous chemokine gra- *Technische Universita¨t Darmstadt, Clemens-Scho¨pf-Institut fur€ Organische Chemie dients formed by diffusion of the protein in agarose gel stamps can und Biochemie, 64287 Darmstadt, Germany; †Technische Universita¨tDarmstadt, Ernst-Berl-Institut fur€ Technische und Makromolekulare Chemie, 64287 Darmstadt, be transferred onto a petri dish surface by stamping. Thus, defined Germany; and ‡Karlsruhe Institute of Technology, Institute of Microstructure Tech- and reproducible protein gradients are obtained and the adhesion, nology, 76344 Eggenstein-Leopoldshafen, Germany activation, formation of lamellipodia, and migration of cells in Received for publication January 21, 2015. Accepted for publication March 31, 2015. response to the immobilized protein can be examined as shown for Address correspondence and reprint requests to Prof. Katja Schmitz, Technische Univer- the example of human neutrophils responding to CXCL8. Therefore, sita¨t Darmstadt, Clemens-Scho¨pf-Institut fur€ Organische Chemie und Biochemie, Alarich- Weiss-Strasse 4, 64287 Darmstadt, Germany. E-mail address: schmitz@biochemie. this setup is suitable for a migration assay based on immobilized tu-darmstadt.de protein to study haptotaxis of leukocytes in response to surface- Abbreviations used in this article: CPI, chemotactic precision index; GAG, glycos- bound chemokine gradients. Agarose stamping of diffusion gra- aminoglycan; FMI, forward migration index in y direction; SMI, sideward migration dients is very simple and inexpensive compared with commercial index in x direction. devices and other established methods like microfluidics, which Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 require complex channel systems and setups. Combined with an

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500148 2 A HAPTOTAXIS ASSAY FOR LEUKOCYTES image processing and statistical evaluation algorithm, our method Isolation of neutrophil granulocytes from human blood yields information about cell behavior at any given time point, and Buffy coats from human blood donors were obtained from the blood donor permits the study of single-cell responses and statistical analysis center of the DRK Frankfurt. Equal volumes of buffy coat, PBS, and 6% of migration. dextran 500 solution were incubated for 30 min at 37˚C. The supernatant was centrifuged for 10 min at 15˚C and 240 3 g. The pellet was washed with PBS and gently resuspended in 10 ml PBS, transferred onto 30 ml Materials and Methods Lymphoprep (Axis-Shield PoC), and centrifuged for 20 min at 4˚C and Expression and purification of CXCL8 600 3 g with the brake turned off. The supernatant was carefully removed and the red pellet was washed with PBS and resuspended in 5 ml PBS. The plasmid pET-22b containing the sequence for CXCL8 was transformed After adding 25 ml deionized water and waiting for 30 s, 17.2 ml of a 3.6% into the expression strain Escherichia coli BL21 (DE3) RIL (45). A sodium chloride solution was added and the cell suspension was centri- modified protocol of Wiese et al. (45) was used for the expression and fuged for 10 min at 240 3 g. The white pellet was washed with 30 ml PBS purification of the protein. The cells were grown in lysogeny broth medium and chemotaxis buffer (RPMI 1640 medium containing 0.2% BSA). with 60 mg/ml ampicillin at 37˚C to an OD600 of 0.6, and the expression was induced with 0.1 mM isopropyl b-D-1-thiogalactopyranoside. After Haptotaxis assay with human neutrophil granulocytes 3 h of expression at 30˚C the cell suspension was centrifuged for 45 min at 4500 rpm and 4˚C. The cell pellet was resuspended in buffer A (40 mM Isolated neutrophils were incubated on the petri dish surface for 30 min at 37˚C. Afterward the cell suspension was removed, and the surface was Na2HPO4, 90 mM NaCl, pH 7.4), supplemented with 1 mM EDTA, 0.2 mg/ml lysozyme, 0.1 mg/ml DNase I, and incubated on ice for 1.5 h. washed with PBS thoroughly and covered with 3 ml chemotaxis buffer. After adding 0.5% Triton X-100, the cell suspension was sonified three The marked area of the gradient was positioned under a phase-contrast 3 times for 30 s (Sonoplus; Bandelin Electronics, Berlin, Germany). After objective. For one experiment an objective with 20 magnification was used, whereas all other experiments were done with a 310 magnification.

incubation with additional DNase I for 30 min at room temperature the Downloaded from ∼ 3 m 2 suspension was centrifuged at 4500 rpm and 4˚C for 45 min. The super- The observable area was 895 671 m . The sample was aligned so that natant was purified via HPLC on an A¨ KTA purifier 10 system (GE the steepest increase of the gradient, determined by the cross sections of Healthcare, Freiburg, Germany) using a 5-ml HiTrap SP FF column (GE the gradient, coincided with the vertical of the screen. The cell behavior Healthcare) with a gradient of 0–30% buffer B (1.5 M NaCl and 40 mM was observed by taking a picture every 60 s for a time span of 3 h. sodium phosphate, pH 7.4) in buffer A. The protein fractions were desalted by a centrifugal filter with a 3000 MWCO membrane (Viaspin 20; Satorius Data analysis Stedim Biotech). Protein concentration was determined by measuring the Cells on the recorded images were tracked using the software ImageJ (46) absorption at 280 nm. with the plugin MTrackJ, and the tracked data were used to generate cell http://www.jimmunol.org/ trajectories for statistical analysis of directionality and speed of cells mi- Production of agarose stamps grating in response to the immobilized chemokine. All further calculations A 3% aqueous solution of agarose (NEEO; Carl Roth GmbH) was heated in were performed with MATLAB (MATLAB and Statistics Toolbox Release a microwave, cast into the cavities of a 384-well plate (Greiner Bio-One), 2012b; The MathWorks, Natick, MA). Because it is difficult to align the and centrifuged for 5 min at 800 3 g. After cooling at 4˚C to harden the microscope perfectly with the gradient, the direction of the gradient may vary by several degrees between individual measurements. To obtain agarose, the stamps were gently pulled from the wells with a forceps and comparable results, in a first step we rotated the obtained cell vectors of cut to a length of 5 mm. The resulting stamping area is ∼3.6 3 3.6 mm. The stamps could be stored in the covered well plates at 4˚C for up to one measurement such that the mean angle of all cell trajectories with y several weeks. respect to the -axis is zero. These modified data were subsequently used

for all statistical calculations. Note that this procedure does not affect the by guest on September 28, 2021 Preparation of protein gradients and stamping procedure migration values; for example, FMI and directness calculated from the data as the exact direction of the gradient are determined this way. The cosu,the A total of 5 ml of a 15 mg/ml protein solution in low-salt PBS was forward migration index in y direction (FMI), and the sideward migration transferred onto a glass slide and the stamp was carefully placed on the index in x direction (SMI) for one single cell were calculated as follows (47): drop so that the long side of the stamp was in contact with the surface. vector of direction of gradient After incubation, the stamp was removed, washed with deionized water, cosu 5 ð1Þ and dried. The stamp was carefully placed into a petri dish (35 3 10 mm; mean vector of migration Cellstar; Greiner Bio-One) with the stamping area, that is, the short site of y 2 y the stamp, facing downward (Fig. 1). Light pressure was applied to im- FMI ¼ end 1 ð2Þ prove the contact, the stamp was incubated for 60 s, and the stamped area li was marked. For the modified protocol, the stamp was treated as described earlier and xend 2 x1 SMI ¼ ð3Þ after soaking with the protein solution completely into the stamp for 60 min, li water was added to the bottom of the stamp. After incubation, the stamp was   washed with deionized water and dried. The stamping procedure was nstep 1=2 2 2 performed as described earlier. with li ¼ + ðxi 2 xi21Þ þðyi 2 yi21Þ ð4Þ i¼2

Detection of protein gradients on surfaces The directness for one single cell has been calculated accordingly: The CXCL8 surfaces were incubated with 10 mg/ml FITC-labeled anti-   1=2 2 2 human CXCL8 (Biolegend) in PBS for 1 h. After washing with PBS, the ðxend 2 x1Þ þðyend 2 y1Þ fluorescent Ab was detected by fluorescence microscopy (Zeiss Observer). directness ¼ ð5Þ For the fluorescence imaging a 32.5 magnification was used that resulted li in a detected area of 3581 3 2683 mm2. The images were analyzed using The values given throughout the text are the mean values over all cells the software ImageJ (46) and the Plugin loci_tools. The relative fluores- within one experiment (12–173 cells) with the SEM for these values. cence intensity across the gradient was calculated and plotted to evaluate For a quantitative analysis of chemotaxis in response to a defined gra- the chemokine cross sections. dient, we propose a chemotactic precision index (CPI), which is discussed in Estimation of the immobilized protein quantity on the surface more detail in the Results and Discussion:

A total of 5 ml CXCL8 solutions in PBS with defined concentrations was FMI2 CPI ¼ cosf FMI ð1 2 jSMIjÞ ¼ ð1 2 jSMIjÞ ð6Þ allowed to dry on a stamped agarose spot on a petri dish surface. The dried directness spots were incubated with 10 mg/ml FITC-labeled anti-human CXCL8 (Biolegend) in PBS for 1 h. After washing, fluorescence was detected by The speed of the cells was calculated for each time step, and the SD was fluorescence microscopy (Zeiss Observer). The fluorescence intensities determined assuming a normal distribution. The Rayleigh test for the were obtained by the software ImageJ (46). After calculating the amount of evaluation of the directed migration was performed as suggested by Moore CXCL8 per area (mm2), a calibration curve was plotted with which the et al. (48). This procedure accounts for different lengths of the cell vectors amount of immobilized CXCL8 on the gradient was estimated. and weights them accordingly, resulting in more accurate values. The Journal of Immunology 3

Results surface and its decrease along the gradient from fluorescence in- Development of diffusion-controlled protein gradients tensities per area after immunostaining. Due to the different size and properties of CXCL8 in comparison For cell migration experiments, continuous protein gradients immo- with BSA and the differences in the agarose used by Mayer et al. bilized noncovalently on a surface had to be created. To generate (44), the optimal diffusion time for CXCL8 within the gel had to be these gradients, we used the method described by Mayer et al. determined. Because of its smaller size we expected shorter diffu- (44), who produced gradients of BSA in an agarose gel by dif- sion times for CXCL8 in comparison with the times of 4–7 h for fusion. These gradients were then transferred by light pressure to BSA reported by Mayer et al. (44). We tested 60, 75, and 90 min for a glass surface functionalized with aldehyde groups for covalent diffusion, and an FITC-labeled anti-CXCL8 Ab was used to detect attachment. This procedure was optimized for our purpose to yield the chemokine under the fluorescence microscope. The obtained gradients of CXCL8 (Fig. 1). To obtain a suitable agarose stamp fluorescence images of the CXCL8 gradients were used to compile for cell experiments in terms of size and smoothness, we used the cross sections of the gradients for comparison of the gradient 384-well plates as a mold for the stamps into which the hot aga- shapes resulting from the different diffusion times (Fig. 2B). rose solution was cast and hardened (Fig 1, step 1). After pulling The stamping of CXCL8 resulted in homogeneous protein the cooled agarose stamps off the well, we obtained cuboids with coatings (Fig. 2A). For all tested diffusion times, the cross section 3 a quadratic base area of 3.6 3.6 mm (Fig. 1, step 2). By incu- of the fluorescence intensity profiles resembled a sigmoidal curve bating the stamp in chemokine solution, a gradient was formed with a plateau on both sides and an approximately linear slope in by diffusion in the gel (Fig. 1, step 3). By gently pushing the the center. Interestingly, the selected diffusion times seemed to stamping area of the agarose on a petri dish surface (Fig. 1, step have no major influence on the inclination of the gradient that Downloaded from 4), the protein could easily be immobilized due to the transfer of ranged between 22 and 23 ng/mm (Table I). For 75- and 90-min a thin layer of agarose without the need for covalent immobili- diffusion time, a linear area of 500 mm between 2000 and 2500 mm zation (Fig. 1, step 5). measured from the edge of the stamp area was identified, and we The polystyrene petri dish surface (Cellstar; Greiner Bio-One) is chose the diffusion time of 75 min for testing cell behavior. functionalized with hydroxyl and carboxyl groups for hydrophi- To obtain a linear gradient over a larger distance, we used a

licity. We assumed that CXCL8 adhered to the negatively charged modified procedure. We soaked the stamp in CXCL8 solution for http://www.jimmunol.org/ surface because of electrostatic interaction of its positive charge at 60 min (Fig. 1, step 3). After adding water to the bottom of the stamp, physiological pH. However, dried CXCL8 from a drop could not be we extended the incubation time to 2 or 3 h (Fig. 2D, 2E). This detected on the petri dish surface after immunostaining, demon- resulted in linear gradients over almost the whole width of the stamp strating that it was not bound on the polystyrene surface. Therefore, between 0 and 3600 mm, but with a more shallow inclination of binding was associated with the agarose, and only the stamping 0.008 or 0.005 ng/mm at 2 or 3 h respectively (Table I). For cell process with the chemokine in the agarose gel resulted in an experiments, we decided to apply the gradient of 2-h incubation time. immobilized protein on the surface. Hence we assume that the To test the homogeneity of the stamped protein surfaces and protein is embedded in the agarose transferred to the surface. the reproducibility of the stamping procedure, we generated five For evaluation of cell migration in response to CXCL8, the stamped surfaces by the use of one single stamp (Fig. 2C). The SDs by guest on September 28, 2021 amount of immobilized CXCL8 on the surfaces was estimated. We of the cross sections of the five gradients were relatively low, prepared a fluorescence intensity calibration curve with defined which indicated good reproducibility. One stamp could be used to amounts of CXCL8 per area detected by a fluorescently labeled produce several immobilized gradients on one or several surfaces. anti-CXCL8 Ab (data not shown). Using this curve, we were able Furthermore, to test the reproducibility of the stamp fabrication to estimate the absolute amount of CXCL8 immobilized on the and the formation of the gradient, we compared three surfaces

FIGURE 1. Schematic description of the stamp fabrication and the stamping procedure. Step 1: hot 3% agarose solution is cast in a 384-well plate, centrifuged, and cooled to solidify the agarose. Step 2: the agarose stamp is gently removed from the wells. Step 3: the stamp is carefully placed on a drop of protein solution so that the long side of the stamp is in contact with the surface. After incubation, the stamp is removed and washed. Step 4: the stamp is turned by 90 degrees and placed with the stamping area facing downward on a petri dish by applying light pressure to improve the contact. Step 5: the stamp is incubated for 60 s on the surface before it is removed. Step 6: cell suspension is added to the surface and the cells are allowed to adhere for 30 min before nonadhering cells are removed. Adherent cells migrating along the protein gradient are observed over 3 h. 4 A HAPTOTAXIS ASSAY FOR LEUKOCYTES Downloaded from

A FIGURE 2. CXCL8 gradients immobilized on a petri dish surface by a stamping procedure. ( ) Fluorescence image of the gradient formed by applying http://www.jimmunol.org/ the parameters of Mayer et al. (44) after 75 min diffusion time. To visualize the protein, FITC-labeled CXCL8 Ab was used. The gradient has a sigmoidal shape with a short linear region. (B) The cross sections of the gradients were determined for 60, 75, and 90 min diffusion time. (D) Fluorescence image of the shallow gradient obtained by the modified method after 2 h diffusion time. To visualize the protein, FITC-labeled CXCL8 Ab was used. (E) The cross sections of the gradients were determined for 2 and 3 h diffusion time. (C and F) Reproducibility of protein stamping procedure. (C) One stamp of 75 min diffusion time of CXCL8 stamped five times. Mean value of five gradients 6 SD. (F) Mean value 6 SD of three independently stamped CXCL8 gradients of 75 min diffusion time using stamps fabricated on 3 different days. stamped with different stamps prepared on different days (Fig. 2F). receptors, CXCR1 and CXCR2, for interaction with the chemokine The SDs were slightly higher compared with the results of repeated CXCL8. Neutrophils show a strong response after activation with by guest on September 28, 2021 stamping but demonstrated the good reproducibility of the whole CXCL8 and are able to migrate along an appropriate gradient with stamp and gradient fabrication procedure. high velocity. These cells are very sensitive, they become activated The protein is immobilized noncovalently by the deposited agarose quickly during the isolation process, and their characteristics are so that some CXCL8 and/or agarose may dissociate and diffuse from dependent on the physical state of the donor (49, 50). Nevertheless, the surface if the protein gradient is covered by cell culture medium neutrophils are widely used for migration studies and provide for several hours during haptotaxis experiments. Therefore, we tested significant results if a sufficient number of independent experi- the stability of the immobilized gradient in medium by evaluating the ments are carried out. fluorescence intensity profiles before and after 3 h of incubation. We first tested neutrophil behavior on the petri dish surface The cross section of the fluorescence intensity after incubation with and on the pure agarose. Over the course of 60 min, neutrophil medium showed a similar curve shape as the untreated CXCL8 granulocytes exhibited the same behavior on the plain petri dish as gradient, but the inclination of the fluorescence intensity gradient on the agarose-coated surface (data not shown). Cells could adhere, decreased to almost half the previous value, from 23 to 13 ng/mm spread over the surface without showing polarization, and show (Fig. 3). To ensure that the loss of CXCL8 on the surface did not activation by formation of lamellipodia. A few cells migrated affect cell behavior and cell migration, we selected the linear region randomly but remained within a maximum radius of 15 mm. The between 1500 and 2500 mm for observation of the cells because in only difference between the two surfaces was that some cells lost this region the gradient was retained during the 3-h experiment. interaction with the agarose surface during the experiment. Experiments with neutrophils were then performed on immo- Response of neutrophil granulocytes to different gradient shapes bilized CXCL8 gradients to ensure that the immobilized amount For haptotaxis experiments, we applied the well-studied cell system of protein was sufficient to activate the cells, to test whether the of primary human neutrophil granulocytes that have two G-coupled conformation of the immobilized protein was appropriate to ac-

Table I. Parameters of the different CXCL8 gradients formed by diffusion

Gradient Diffusion Time CXCL8 (ng/mm2) Increase of CXCL8 (ng/mm) Linear Area (mm) 1 60 min 13.5 to 4.6 22.3 2200–2600 75 min 16.2 to 4.6 23.1 2000–2500 90 min 15.9 to 4.9 22.0 2000–2500 22h.20 to 2.9 0.008 0–3600 3h .20 to 5.1 0.005 0–3600 Gradient 1 was formed by the method of Mayer et al. (44) and gradient 2 by the modified protocol. Total amounts of immobilized CXCL8 were determined, as well as the inclinations and distances of the linear areas of the gradients. The Journal of Immunology 5

be suitable to induce migration of neutrophil granulocytes and was chosen for the following haptotaxis experiments.

Statistical analysis of haptotactic behavior Three independent cell experiments were conducted to evaluate haptotaxis of the cells. Cell responses were observed over a time span of 3 h. The linear region of the gradient between 2000 and 2500 mm was observed. The initial parameters, consisting of an observed area of 448 3 336 mm2 and time intervals of 5 min between every frame (Fig. 5C), proved to be not ideal. The mi- gration could not be followed over the whole time of 3 h because FIGURE 3. Stability of the immobilized CXCL8 gradient after 3 h in- cubation in cell culture medium. (A) Fluorescence image of FITC-labeled of migration out of the visual field of several migrating cells. CXCL8 Ab to visualize the CXCL8 gradient stamped on a petri dish. (B) Therefore, in the following independent experiments, a surface 2 Cross section of the gradient before and after incubation in medium. Solid area of 895 3 671 mm was observed with time intervals of 60 s line represents CXCL8 gradient after 75 min diffusion time before the over 3 h. This procedure resulted in a better resolution of the incubation with medium for 3 h. Dashed line represents the remaining migrating cells (Fig. 5D, 5E). CXCL8 gradient after 3-h incubation in medium. After rotating the obtained cell vectors, the cell trajectories from all experiments indicated a directed migration along the CXCL8 tivate its receptors, and to determine whether the protein distri- gradient that proceeded along the y-axis. It is noticeable that the Downloaded from bution would be recognized by the cells as a gradient. On the paths of the migrating cells toward higher CXCL8 amounts were shallow CXCL8 gradient built by the modified procedure (Fig. 2E), very straight and hardly any haptokinesis was observable (Fig. 5). the cells adhered and showed activation by the formation of The Rayleigh test (48) for the evaluation of the migration vectors lamellipodia, but no different cell behavior in comparison with the in uniform circular distribution indicated levels of significance for petri dish or agarose surface was observable (data not shown). all experiments, with p , 0.001 corresponding to a specific mi-

Observation of the cells adhering to the sigmoidal gradient built gration along one direction. http://www.jimmunol.org/ by the method of Mayer et al. (44) revealed that the number of The migrating fractions of adherent cells lay between 30% for adherent cells depends on the amount of CXCL8 on the surface the first two and 50% for the third experiment. Nonmigrating cells (Fig. 4). On the plateaus of the gradient, no directed cell migration adhered to the surface, but they were uniformly distributed and not could be observed, but the activation was visible by the formation polarized (Fig. 5B). They were tethered to the petri dish surface of lamellipodia. This may be explained by the shallow concen- without any observable migration. In comparison with the ad- tration gradient in these regions or too high or too low CXCL8 herent cells on the petri dish surface, the nonmigrating cells were concentrations for induction of migration. It is well-known that more spherical and less spread out, illustrated by the size of the cells chemotaxis is dose dependent and follows a bell-shaped curve (Fig. 5A, 5B). The migrating cells were polarized and showed whereby the cells migrate less at low or high concentrations of a leading edge consisting of lamellipodia followed by the cell body by guest on September 28, 2021 CXCL8 (51, 52). In the area of the linear slope, migrating neu- and a terminal uropod (Fig. 5B). trophils could be observed. Therefore, the immobilized sigmoidal For statistical analysis of chemotaxis, diverse chemotactic in- CXCL8 gradient formed by a diffusion time of 75 min proved to dices are presented in the literature based on the assumption that

FIGURE 4. Cell adherence on the sigmoidal CXCL8 gradient. (A) Neutrophil granulocytes recognized the surface-bound CXCL8 gradient, and the cell count increases with increasing CXCL8 amount. Original magnification 316. Scale bar, 100 mm. (B) Cross section of the CXCL8 gradient from fluorescence image. (C) Mean of the cell count per image 6 SEM (n =3). 6 A HAPTOTAXIS ASSAY FOR LEUKOCYTES

FIGURE 5. Neutrophil images and migration trajectories of the hapto- taxis experiments. (A) Adherent neutrophils on an untreated petri dish surface. Cells are well spread without polarization. Scale bar, 10 mm. (B) FIGURE 6. Chemotaxis indices. Vector image to illustrate the parameter Downloaded from Migrating and nonmigrating neutrophils on a CXCL8 gradient. Migrating for the different calculations with the equations for the calculation of the u cells are polarized with a leading edge consisting of lamellipodia followed cos , FMI, SMI, directness, and the CPI. Gray lines indicate migration by the cell body and a terminal uropod. Nonmigrating cells are uniformly paths of cells. distributed and not polarized. The CXCL8 concentration is increasing from m C E below. Scale bar, 10 m. ( – ) Migration trajectories of three independent of FMI together with low SMI values perpendicular to the gradient experiments. The direction of the gradient is represented by the triangle

demonstrate highly directed migration in response to a given http://www.jimmunol.org/ pointing toward the higher concentration of CXCL8. The experiments were performed with freshly isolated human neutrophils. chemoattractant gradient. The FMI and the directness contain important information, but not the precision by which the cells follow the direction of a given gradient. the observed phenomenon is chemotaxis rather than chemokinesis. The chemotaxis coefficient based on the Keller and Segel model The evaluation of haptotaxis occurs equivalent to chemotaxis as for chemotaxis is much more detailed because it includes the generally two-dimensional systems are examined. concentration of the chemoattractant, the gradient steepness, and Agrawal et al. (49) and Tharp et al. (53) described a chemotaxis other cell-dependent parameters by which chemotaxis can be index that results from the cosine of the angle u between the di- described (59, 60). The coefficient is useful but very specific. Due rection of the gradient and the mean vector of migration (Fig. 6, to its dependence on the exact experimental setup it cannot be by guest on September 28, 2021 Eq. 1). This value ranges from 21 to +1, whereby +1 indicates used to compare chemotaxis in different setups. perfect migration along the gradient (54, 55). This index exhibits To compare the chemotactic precision, we propose a CPI as the two disadvantages. First, the direction of the gradient has to be product of the cosine of the angle between the vectors of FMI and known exactly to avoid major errors. Second, the evaluation of directness with the FMI and one minus the absolute value of the chemotaxis occurs on the basis of a small angle. If there is less SMI (Fig. 6, Eq. 6). The cosine of the angle f specifies whether migration along the y direction, corresponding to the gradient the obtained data are a result of chemotaxis as small angles rep- direction, a small value for u will result as well and the index will resent directed migration along the gradient. This angle is multi- incorrectly indicate strong chemotaxis. In addition, in the case of plied by the FMI, which gives information about how strong random migration, that is, chemokinesis, the average of this angle migration along the gradient is. By additional multiplication of will approach 0 and consequently cos u will approach 1. Taken one minus the absolute value of SMI, information about migration together, this value is neither suitable to decide whether there is in x direction is included in the CPI. The angle between the FMI chemotaxis nor can it be used for quantitative analysis. and the directness could be replaced by the angle u used in the Related problems occur with the FMI (Fig. 6, Eq. 2) (28, 47, 56, literature if the direction of the gradient is exactly known (54, 55). 57). This index is simply a measure of the migration along the In the case of chemokinesis, only random movements are ob- gradient and disregards the migration in the perpendicular direc- served and the angle may adopt any value, whereas the FMI will tion. Another approach uses the directness as a measure of che- exhibit a low value and the CPI will be low as well. motaxis that considers only directed migration (Fig. 6, Eq. 5). Possible limiting cases are provided to illustrate the weakness of Thus, it bears these problems like the FMI with the disregard of the directness and the FMI as chemotaxis indices in comparison the x direction of migration as well (58). Directness weights the with the CPI (Fig. 7B). Case 1 shows a directness of 1 with an FMI migration in x and y direction equally, and only information about of 0.2 that results in an SMI of 0.98 and a CPI of 0.0008. This case the ratio of the path length in x and y direction is considered, shows that directness alone is not sufficient to draw conclusions whereas the overall direction of migration itself is not examined. about chemotaxis, because the main direction of migration dis- In the case of completely linear movement along the gradient, tinctively continues along the axis of abscissae, perpendicular to both FMI and directness are 1. However, high values of FMI in the the gradient. It is further shown that an FMI of 0.2 (as published range of 0.7 do not sufficiently prove migration along the gradient. by Lin et al. [28]) does not indicate chemotaxis, but neither For example, an FMI of 0.7 and a value of 1 for directness are excludes it. Nevertheless it is not possible to quantify chemotaxis obtained for a high value of 0.7 for the SMI in x direction. Hence by utilization of the directness or the FMI. In case 2, a directness the direction of the migration would not be along the gradient, but of 1 with an FMI of 0.7 is shown, which results in an SMI of 0.7. along a path in a 45˚ angle to the gradient. Accordingly, the mi- In this case, a preferred direction of migration exists, but this gration in x direction has to be included as well. Only high values direction is rotated by 45˚ rather than the gradient direction itself. The Journal of Immunology 7

FIGURE 7. Parameters for migration. (A) Obtained values for direct- ness, FMI, SMI, and the CPI. (B) Possible limiting cases for quantifying chemotaxis by using the directness, the FMI, and the SMI in comparison with the CPI. Downloaded from

The directness and FMI both have high values; however, there is no chemotaxis along the gradient, as indicated by a CPI of 0.14. FIGURE 8. Histograms of average migration speed. (A–C) Histograms Case 3 indicates that low values of directness result in low values of the speed measured in three independent experiments. (D) Speed of the for the FMI and subsequently also for SMI. Therefore, the CPI is neutrophils during the first, second, and third hour of migration and the very low, classifying cell movement as no chemotaxis. In case 4, 6 mean speeds SD of all data points of the experiments were determined. http://www.jimmunol.org/ the directness has a value of 0.35, as in an example reported by Sackmann et al. (58). An FMI of 0.3 results in an SMI of 0.18, a short linear region. By extending the linear region, the inclina- which leads to a low CPI of 0.21. These data reflect the limits of tion became shallower. Thus, the shape of the gradient can be CPI. In this case, directed migration is present, but it is too weak varied via the incubation protocol. However, the variability of the to produce satisfactory results in a chemotaxis assay and the CPI printable shape method is limited. Microfluidic devices are more as well. Case 5 confirms the limits of the CPI as weak chemotaxis versatile because they allow building almost any shape of soluble leads to low values for the CPI. With a directness of 0.2 and an gradient in which the linear region and the inclination can be FMI of 0.2 (SMI is 0), the CPI has just a value of 0.2. accurately adjusted (25, 26, 60). However, designing and manu- Calculating these values for our experiments provides values for facturing the respective gradient generator in micrometer scale may by guest on September 28, 2021 the directness in the range of 0.66 to 0.78, clearly indicating be time consuming and requires specialized equipment and expe- a preferred direction of migration (Fig. 7A). Likewise, the high rience, whereas the diffusion gradient stamping method is very values for FMI in the range of 0.64 to 0.75 clearly emphasize simple and inexpensive in processing. In particular, there is no directed migration along the gradient. For the SMI, we obtained need for special equipment. As opposed to classical methods like very low values between 0.03 and 0.06. Using the values for di- the Boyden (20), Zigmond (21), and Dunn chambers (22), the rectness FMI and SMI, the CPI was calculated by Eq. 6 (Fig. 6). stamping method leads to defined immobilized gradients that are For the first experiment with only 12 cells a value of 0.68 was reproducible in shape. The temporal and spatial stability is not obtained, whereas the values for the second experiment with 31 entirely ensured as the CXCL8 amount on the surface decreases cells was 0.71 and for the third with 173 cells was 0.59. Due to with exposure to medium over 3 h. Although the shape of the similar values of directness and FMI for the experiments, it is not gradient remains similar, the absolute amounts of CXCL8 decrease. surprising that the values obtained for the CPI do not differ much The diffusion could possibly lead to a soluble gradient in close from each other. proximity to the surface and in this way exert influence on the Analysis of speed distribution activation and migration of the cells. In vivo the situation is similar as the cells are exposed to an immobilized gradient, as well as to The speed of neutrophils provides information about the magnitude a soluble gradient (10, 61). Cells exposed to these surfaces recog- of chemotaxis/haptotaxis. Therefore, the speed distributions of the nize these gradients and do not alter their velocity as absolute cell migration experiments were compiled (Fig. 8). The mean CXCL8 amounts on the surface decrease while the gradient is m speed of the cells lay between 3 and 4 m/min, whereas the range maintained. m of the speed was between 0 and a maximum of 10 m/min. The high inclination of the gradient obtained by using the method During the whole experiment no significant change in the aver- of Mayer et al. (44) seemed to be very important for cell behavior age speed over time was observed (Fig. 8D), so the cells were because the shallow gradient with an inclination of 0.008 ng/mm apparently not affected by the decrease of the CXCL8 amount was not able to elicit a response in neutrophils different from the described earlier. Therefore, sufficient CXCL8 remained on the uncoated surface. It is known that the neutrophil migration is de- surface for activating neutrophils. pendent on the inclination of the gradient and the CXCL8 concen- tration: with microfluidic channels, a concentration range between Discussion 0 and 5–500 ng/ml over a linear gradient region of 500 mmledto We applied the method from Mayer et al. (44) to build protein CXCL8-induced neutrophil migration (53, 60). Thereby a range of gradients in agarose gels to form surface-bound chemokine gra- 0 to 50 ng/ml equates to an inclination of 100 ng/mm on a gradient of dients. With adjusted parameters, gradients with defined, repro- 500-mm length. The inclination of 23 ng/mm obtained by our method ducible shape were generated. We obtained a steep inclination but is roughly one quarter of the inclination obtained by microfluidics 8 A HAPTOTAXIS ASSAY FOR LEUKOCYTES that led to chemotaxis of neutrophils (60), but it was sufficient to 3 and 4 mm/min, and these results were in agreement with the induce migration as opposed to the shallow gradients with an incli- literature, which describes average values of 2.5 mm/min (66) and nation of 0.008 ng/mm (55). The immobilization strategy mimics the speeds between 2 and 10 mm/min depending on the slope of the in vivo situation of leukocyte migrationinresponsetosurface-bound gradient and the CXCL8 amount (28, 53, 60, 62). chemokines as the chemokine is immobilized via GAGs on endo- In summary, we have demonstrated a very simple method for thelial cells. studying haptotaxis of leukocytes in response to surface-bound Notably, only a part of 30 to 50% of the cells migrated along the chemokine gradients. The transfer of diffusion gradients in aga- gradient, whereas the rest were still viable but without polarization rose gels to surfaces works well for the chemokine CXCL8. By (Fig. 5A). The cells showed activation of CXCL8 by formation of using time-lapse microscopy, information on the single-cell level of lamellipodia but in its spherical morphology. The CXCL8 gradient a large number of cells can be gathered easily and statistical data could not induce cell migration in these cells or the cells were not about this cell population can be derived. Only small amounts of able to recognize the gradient. In the first reported chemotaxis cells, protein, and other reagents are needed, making this assay easy experiment with CXCL8 in a multiwell chamber assay, Yoshimura and inexpensive to perform. et al. (5) already observed that just 54% of the neutrophils mi- The assay is very versatile and gives the opportunity to test the grated at the optimal CXCL8 concentration of 10 nM. Keenan influence of almost every protein gradient immobilized onto a et al. (62) made similar observations on cell culture dish surfaces surface to cells and their behavior. The correlation of migratory where a significant part of 38% of all cells showed no migration cell behavior with the gradient shape or protein concentration and but changing and formation of lamellipodia. This could surface modifications can be investigated, as well as changes in cell be an indication that the surface influences the cells in a way that behavior in the presence of inhibitors. In addition, the influence of Downloaded from prevents some cells for migrating by forming a stronger binding soluble chemoattractants on cells following surface-bound gra- between the cell and the surface. In transwell assays to study the dients can be studied (29, 57, 67). The presented model system chemotaxis of neutrophils in response to CXCL8, we obtained of neutrophils responding to CXCL8 is only one example of the similar results. The fraction of migrating cells lay between 30 and wide field of chemokines guiding the migration of immune cells. 70%. The reason for the nonmigrating cells is unclear, but there are Because chemokines are not the only determinant of immune cell

some possible causes. We assumed that the sensitivity and quick migration, the role of other chemoattractants like complement http://www.jimmunol.org/ activation of the neutrophils, as well as the physical state of the factor 5a (68), inflammatory cytokines such as TNF (69), growth donor, determine how many cells are susceptible to CXCL8 (49, 50). factors like CSF-1 (68), or modulating factors such as adhesion Furthermore, adhesion proteins play an important role and mediate molecules like selectins (70) or integrins (64) could be investigated leukocyte migration (63, 64). The influence of adhesive ligands on with this system. The directed migration of cells is involved in in- cell migration depends sensitively on several integrin–ligand inter- flammation, development of the immune system, survival strategies actions like ligand levels, integrin levels, and their binding affinities of pathogens, and allergic reactions. Therefore, an easy method to (65). By the integration of adhesive ligands on the gradient surface study immobilized gradients of individual proteins in vitro is a useful of our assay, a larger fraction of cells might be able to migrate and tool to better understand their individual impact or to confirm hy- this issue will be a matter of further research. potheses derived from in vivo observations. In gradients produced by by guest on September 28, 2021 For statistical analysis of haptotaxis, migration trajectories were this method, the proteins are immobilized by noncovalent interactions compiled and a chemotaxis index for the evaluation had to be selected. without any modifications so that the method can be performed with Evaluation of commonly used indices like the angle u, the directness, recombinant proteins, as well as with native proteins purified from and the FMI showed that these indices could not give quantitative tissues or body fluids. Another interesting aspect of this assay information about the precision of chemotaxis, because they do not system is the possibility of studying the interplay of surface-bound take all parameters of migration into account (see Fig. 7B). There- protein gradients and soluble factors. Only a few years ago, reports fore, we introduce an alternative, the CPI for the evaluation of che- have pointed out the different effects triggered by surface-bound motaxis. The CPI includes all necessary information of migration: protein gradients and soluble gradients (71), and by now this phe- the angle w between the directness and the direction of the gradient, nomenon has only been investigated for a limited number of model as well as the FMI and the SMI. For our experiments, we obtained systems. A lot of research still needs to be done, and a straightfor- CPI values $0.6, indicating highly precise migration in the direction ward and inexpensive method to produce surface-bound gradients of the gradient and showing that the developed haptotactic assay with unmodified proteins will add to the progress in this area. poses a powerful tool to investigate haptotaxis. Finally, the proposed CPI is suitable for quantification of cell Our CPI bears several advantages over the indices reported in migration, chemotaxis, and haptotaxis. It is independent of the ap- the literature. First, the value is simple to calculate based on the plication as long as the migration data can be recorded on a single-cell established chemotaxis indices: directness, FMI, and SMI. Second, level in a time-resolved manner. It indicates the precision by which the resulting value between 0 and 1 allows an intuitive evaluation of cells follow the direction of a given gradient of chemotaxis much the precision by which cells follow the direction of a given gradient; better than the standard values used in the literature. high values indicate strong and precise chemotaxis, whereas low values show weak chemotaxis and may hint chemokinesis. Third, Acknowledgments the index is independent of the underlying chemotaxis assay if the We thank Anke Imrich for protein expression and Marina Joest and Kevin obtained data permit the calculation of the index. Nevertheless, Brahm for scientific discussion. the CPI has its limits: the case of weak migratory responses, when the readout of a migration assay may not be meaningful and an alternative assay should be used. Disclosures In addition, the speed of the cell migration during the first, The authors have no financial conflicts of interest. second, and third hour of the experiment was considered, as well as the speed over the whole experiment time span. Frevert et al. References (66) reported similar speed distributions for neutrophils in a 1. Baggiolini, M. 2001. Chemokines in pathology and medicine. J. Intern. Med. range up to 10 mm/min. The mean speed of the cells lay between 250: 91–104. The Journal of Immunology 9

2. Proudfoot, A. E. 2002. Chemokine receptors: multifaceted therapeutic targets. 31. Rot, A. 1993. Neutrophil attractant/activation protein-1 (interleukin-8) induces Nat. Rev. Immunol. 2: 106–115. in vitro neutrophil migration by haptotactic mechanism. Eur. J. Immunol. 23: 3. Gerard, C., and B. J. Rollins. 2001. Chemokines and disease. Nat. Immunol. 2: 303–306. 108–115. 32. Liedberg, B., and P. Tengvall. 1995. Molecular gradients of. omega.-substituted 4. Mantovani, A., M. A. Cassatella, C. Costantini, and S. Jaillon. 2011. Neutrophils alkanethiols on gold: preparation and characterization. Langmuir 11: 3821– in the activation and regulation of innate and adaptive immunity. Nat. Rev. 3827. Immunol. 11: 519–531. 33. Terrill, R. H., K. M. Balss, Y. Zhang, and P. W. Bohn. 2000. Dynamic monolayer 5. Yoshimura, T., K. Matsushima, S. Tanaka, E. A. Robinson, E. Appella, gradients: active spatiotemporal control of alkanethiol coatings on thin gold J. J. Oppenheim, and E. J. Leonard. 1987. Purification of a human - films. J. Am. Chem. Soc. 122: 988–989. derived neutrophil chemotactic factor that has peptide sequence similarity to 34. von Philipsborn, A. C., S. Lang, A. Bernard, J. Loeschinger, C. David, other host defense cytokines. Proc. Natl. Acad. Sci. USA 84: 9233–9237. D. Lehnert, M. Bastmeyer, and F. Bonhoeffer. 2006. Microcontact printing of 6. Baggiolini, M., A. Walz, and S. L. Kunkel. 1989. Neutrophil-activating peptide- guidance molecules for generation of graded patterns. Nat. Protoc. 1: 1/interleukin 8, a novel cytokine that activates neutrophils. J. Clin. Invest. 84: 1322–1328. 1045–1049. 35. Lehnert, D., B. Wehrle-Haller, C. David, U. Weiland, C. Ballestrem, 7. Holmes, W. E., J. Lee, W.-J. Kuang, G. C. Rice, and W. I. Wood. 1991. Structure and B. A. Imhof, and M. Bastmeyer. 2004. Cell behaviour on micropatterned sub- functional expression of a human interleukin-8 receptor. Science 253: 1278–1280. strata: limits of geometry for spreading and adhesion. J. Cell 8. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. He´bert, R. Horuk, Sci. 117: 41–52. K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power. 2000. Inter- 36. von Philipsborn, A. C., S. Lang, J. Loeschinger, A. Bernard, C. David, national union of pharmacology. XXII. Nomenclature for chemokine receptors. D. Lehnert, F. Bonhoeffer, and M. Bastmeyer. 2006. Growth cone navigation in Pharmacol. Rev. 52: 145–176. substrate-bound ephrin gradients. Development 133: 2487–2495. 9. Samanta, A. K., J. J. Oppenheim, and K. Matsushima. 1990. Interleukin 8 37. Hjortø, G. M., M. Hansen, N. B. Larsen, and T. N. Kledal. 2009. Generating (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own substrate bound functional chemokine gradients in vitro. Biomaterials 30: 5305– receptor expression on human neutrophils. J. Biol. Chem. 265: 183–189. 5311. 10. Rot, A. 1992. Endothelial cell binding of NAP-1/IL-8: role in neutrophil emi- 38. Dominguez, G. A., and D. A. Hammer. 2014. Effect of adhesion and chemokine gration. Immunol. Today 13: 291–294.

presentation on T-lymphocyte haptokinesis. Integr Biol (Camb) 6: 862–873. Downloaded from 11. Middleton, J., S. Neil, J. Wintle, I. Clark-Lewis, H. Moore, C. Lam, M. Auer, 39. Delamarche, E., A. Bernard, H. Schmid, B. Michel, and H. Biebuyck. 1997. E. Hub, and A. Rot. 1997. Transcytosis and surface presentation of IL-8 by Patterned delivery of immunoglobulins to surfaces using microfluidic networks. venular endothelial cells. Cell 91: 385–395. Science 276: 779–781. 12. Lawrence, M. B., and T. A. Springer. 1991. Leukocytes roll on a selectin at 40. Delamarche, E., A. Bernard, H. Schmid, A. Bietsch, B. Michel, and physiologic flow rates: distinction from and prerequisite for adhesion through H. Biebuyck. 1998. Microfluidic networks for chemical patterning of substrates: integrins. Cell 65: 859–873. design and application to bioassays. J. Am. Chem. Soc. 120: 500–508. 13. Handel, T. M., Z. Johnson, S. E. Crown, E. K. Lau, A. E. Proudfoot, and 41. Juncker, D., H. Schmid, A. Bernard, I. Caelen, B. Michel, N. de Rooij, and A. E. Proudfoot. 2005. Regulation of protein function by glycosaminoglycans— E. Delamarche. 2001. Soft and rigid two-level microfluidic networks for pat- as exemplified by chemokines. Annu. Rev. Biochem. 74: 385–410. terning surfaces. J. Micromech. Microeng. 11: 532–541. http://www.jimmunol.org/ 14. Harada, A., N. Sekido, T. Akahoshi, T. Wada, N. Mukaida, and K. Matsushima. 42. Martin, B. D., S. L. Brandow, W. J. Dressick, and T. L. Schull. 2000. Fabrication J. 1994. Essential involvement of interleukin-8 (IL-8) in acute inflammation. and application of hydrogel stampers for physisorptive microcontact printing. Leukoc. Biol. 56: 559–564. Langmuir 16: 9944–9946. 15. Brennan, F. M., C. O. Zachariae, D. Chantry, C. G. Larsen, M. Turner, 43. Brandow, S. L., T. L. Schull, B. D. Martin, D. C. Guerin, and W. J. Dressick. R. N. Maini, K. Matsushima, and M. Feldmann. 1990. Detection of interleukin 8 2002. Use of low-temperature thermal alkylation to eliminate ink migration in biological activity in synovial fluids from patients with and microcontact printed patterns. Chemistry 8: 5363–5367. production of interleukin 8 mRNA by isolated synovial cells. Eur. J. Immunol. 44. Mayer, M., J. Yang, I. Gitlin, D. H. Gracias, and G. M. Whitesides. 2004. 20: 2141–2144. Micropatterned agarose gels for stamping arrays of proteins and gradients of 16. Kraan, M. C., D. D. Patel, J. J. Haringman, M. D. Smith, H. Weedon, proteins. Proteomics 4: 2366–2376. M. J. Ahern, F. C. Breedveld, and P. P. Tak. 2001. The development of clinical 45. Wiese, D., and K. Schmitz. 2011. Expression of recombinant human interleukin- signs of rheumatoid synovial inflammation is associated with increased synthesis

8 and its purification using a single buffer system. J. Immunol. Methods 364: 77– by guest on September 28, 2021 of the chemokine CXCL8 (interleukin-8). Arthritis Res. 3: 65–71. 82. 17. Strassburg, A., D. Droemann, G. van Zandbergen, H. Kothe, and K. Dalhoff. 46. Schneider, C. A., W. S. Rasband, and K. W. Eliceiri. 2012. NIH Image to 2004. Enhanced PMN response in chronic bronchitis and community-acquired ImageJ: 25 years of image analysis. Nat. Methods 9: 671–675. pneumonia. Eur. Respir. J. 24: 772–778. 47. Foxman, E. F., E. J. Kunkel, and E. C. Butcher. 1999. Integrating conflicting 18. Lund, B. T., N. Ashikian, H. Q. Ta, Y. Chakryan, K. Manoukian, S. Groshen, chemotactic signals. The role of memory in leukocyte navigation. J. Cell Biol. W. Gilmore, G. S. Cheema, W. Stohl, M. E. Burnett, et al. 2004. Increased CXCL8 (IL-8) expression in Multiple Sclerosis. J. Neuroimmunol. 155: 161– 147: 577–588. 48. Moore, B. R. 1980. A modification of the Rayleigh test for vector data. Bio- 171. 19. Gijsbers, K., G. Van Assche, S. Joossens, S. Struyf, P. Proost, P. Rutgeerts, metrika 67: 175–180. K. Geboes, and J. Van Damme. 2004. CXCR1-binding chemokines in inflam- 49. Agrawal, N., M. Toner, and D. Irimia. 2008. Neutrophil migration assay from matory bowel diseases: down-regulated IL-8/CXCL8 production by leukocytes a drop of blood. Lab Chip 8: 2054–2061. in Crohn’s disease and selective GCP-2/CXCL6 expression in inflamed intestinal 50. Hoang, A. N., C. N. Jones, L. Dimisko, B. Hamza, J. Martel, N. Kojic, and tissue. Eur. J. Immunol. 34: 1992–2000. D. Irimia. 2013. Measuring neutrophil speed and directionality during chemo- 20. Boyden, S. 1962. The chemotactic effect of mixtures of antibody and antigen on taxis, directly from a droplet of whole blood. Technology (Singap World Sci) 1: polymorphonuclear leucocytes. J. Exp. Med. 115: 453–466. 49–57. 21. Zigmond, S. H. 1977. Ability of polymorphonuclear leukocytes to orient in 51. Webb, L. M., M. U. Ehrengruber, I. Clark-Lewis, M. Baggiolini, and A. Rot. gradients of chemotactic factors. J. Cell Biol. 75: 606–616. 1993. Binding to heparan sulfate or heparin enhances neutrophil responses to 22. Zicha, D., G. A. Dunn, and A. F. Brown. 1991. A new direct-viewing chemotaxis interleukin 8. Proc. Natl. Acad. Sci. USA 90: 7158–7162. chamber. J. Cell Sci. 99: 769–775. 52. Foxman, E. F., J. J. Campbell, and E. C. Butcher. 1997. Multistep navigation and 23. Dahlgren, C., K.-E. Magnusson, and T. Sundqvist. 1984. Concentration gradients the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139: 1349–1360. in the under-agarose chemotaxis assay system with attractant surface adsorption. 53. Tharp, W. G., R. Yadav, D. Irimia, A. Upadhyaya, A. Samadani, O. Hurtado, J. Immunol. Methods 75: 23–29. S. Y. Liu, S. Munisamy, D. M. Brainard, M. J. Mahon, et al. 2006. Neutrophil 24. Lin, F., W. Saadi, S. W. Rhee, S.-J. Wang, S. Mittal, and N. L. Jeon. 2004. chemorepulsion in defined interleukin-8 gradients in vitro and in vivo. J. Leukoc. Generation of dynamic temporal and spatial concentration gradients using Biol. 79: 539–554. microfluidic devices. Lab Chip 4: 164–167. 54. Sapey, E., H. Greenwood, G. Walton, E. Mann, A. Love, N. Aaronson, 25. Jeon, N. L., S. K. Dertinger, D. T. Chiu, I. S. Choi, A. D. Stroock, and R. H. Insall, R. A. Stockley, and J. M. Lord. 2014. Phosphoinositide 3-kinase G. M. Whitesides. 2000. Generation of solution and surface gradients using inhibition restores neutrophil accuracy in the elderly: toward targeted treatments microfluidic systems. Langmuir 16: 8311–8316. for immunosenescence. Blood 123: 239–248. 26. Dertinger, S. K., D. T. Chiu, N. L. Jeon, and G. M. Whitesides. 2001. Generation 55. Chen, L., C. Janetopoulos, Y. E. Huang, M. Iijima, J. Borleis, and of gradients having complex shapes using microfluidic networks. Anal. Chem. P. N. Devreotes. 2003. Two phases of polymerization display different 73: 1240–1246. dependencies on PI(3,4,5)P3 accumulation and have unique roles during che- 27. Dertinger, S. K. W., X. Jiang, Z. Li, V. N. Murthy, and G. M. Whitesides. 2002. motaxis. Mol. Biol. Cell 14: 5028–5037. Gradients of substrate-bound laminin orient axonal specification of neurons. 56. Wu, J., L. Ouyang, N. Wadhawan, J. Li, M. Zhang, S. Liao, D. Levin, and F. Lin. Proc. Natl. Acad. Sci. USA 99: 12542–12547. 2014. A compact microfluidic system for cell migration studies. Biomed. 28. Lin, F., C. M.-C. Nguyen, S.-J. Wang, W. Saadi, S. P. Gross, and N. L. Jeon. Microdevices 16: 521–528. 2004. Effective neutrophil chemotaxis is strongly influenced by mean IL-8 57. Byrne, M. B., Y. Kimura, A. Kapoor, Y. He, K. S. Mattam, K. M. Hasan, concentration. Biochem. Biophys. Res. Commun. 319: 576–581. L. N. Olson, F. Wang, P. J. Kenis, and C. V. Rao. 2014. Oscillatory behavior of 29. Lin, F., C. M.-C. Nguyen, S.-J. Wang, W. Saadi, S. P. Gross, and N. L. Jeon. neutrophils under opposing chemoattractant gradients supports a winner-take-all 2005. Neutrophil migration in opposing chemoattractant gradients using mechanism. PLoS ONE 9: e85726. Available at: http://journals.plos.org/plosone/ microfluidic chemotaxis devices. Ann. Biomed. Eng. 33: 475–482. article?id=10.1371/journal.pone.0085726. 30. Kim, B. J., and M. Wu. 2012. Microfluidics for mammalian cell chemotaxis. 58. Sackmann, E. K.-H., E. Berthier, E. A. Schwantes, P. S. Fichtinger, M. D. Evans, Ann. Biomed. Eng. 40: 1316–1327. L. L. Dziadzio, A. Huttenlocher, S. K. Mathur, and D. J. Beebe. 2014. Char- 10 A HAPTOTAXIS ASSAY FOR LEUKOCYTES

acterizing asthma from a drop of blood using neutrophil chemotaxis. Proc. Natl. 66. Frevert, C. W., G. Boggy, T. M. Keenan, and A. Folch. 2006. Measurement of Acad. Sci. USA 111: 5813–5818. cell migration in response to an evolving radial chemokine gradient triggered by 59. Keller, E. F., and L. A. Segel. 1971. Model for chemotaxis. J. Theor. Biol. 30: 225–234. a microvalve. Lab Chip 6: 849–856. 60. Li Jeon, N., H. Baskaran, S. K. W. Dertinger, G. M. Whitesides, L. Van de Water, 67. Heit, B., S. Tavener, E. Raharjo, and P. Kubes. 2002. An intracellular signaling and M. Toner. 2002. Neutrophil chemotaxis in linear and complex gradients of hierarchy determines direction of migration in opposing chemotactic gradients. interleukin-8 formed in a microfabricated device. Nat. Biotechnol. 20: 826–830. J. Cell Biol. 159: 91–102. 61. Snyderman, R., and E. J. Goetzl. 1981. Molecular and cellular mechanisms of 68. Webb, S. E., J. W. Pollard, and G. E. Jones. 1996. Direct observation and leukocyte chemotaxis. Science 213: 830–837. quantification of macrophage chemoattraction to the growth factor CSF-1. J. Cell 62. Keenan, T. M., C. W. Frevert, A. Wu, V. Wong, and A. Folch. 2010. A new Sci. 109: 793–803. method for studying gradient-induced neutrophil desensitization based on an 69. Wang, J. M., S. Walter, and A. Mantovani. 1990. Re-evaluation of the chemotactic open microfluidic chamber. Lab Chip 10: 116–122. activity of tumour necrosis factor for . Immunology 71: 364–367. 63. Carlos, T. M., and J. M. Harlan. 1994. Leukocyte-endothelial adhesion mole- 70. Subramanian, H., J. J. Grailer, K. C. Ohlrich, A. L. Rymaszewski, J. J. Loppnow, cules. Blood 84: 2068–2101. M. Kodera, R. M. Conway, and D. A. Steeber. 2012. Signaling through 64. Lishko, V. K., V. P. Yakubenko, and T. P. Ugarova. 2003. The interplay between L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary integrins alphaMbeta2 and a5b1 during cell migration to fibronectin. Exp. Cell lymphoid tissue chemokine. J. Immunol. 188: 3223–3236. Res. 283: 116–126. 71. Schumann, K., T. La¨mmermann, M. Bruckner, D. F. Legler, J. Polleux, 65. Palecek, S. P., J. C. Loftus, M. H. Ginsberg, D. A. Lauffenburger, and J. P. Spatz, G. Schuler, R. Fo¨rster, M. B. Lutz, L. Sorokin, and M. Sixt. 2010. A. F. Horwitz. 1997. Integrin-ligand binding properties govern cell migration Immobilized chemokine fields and soluble chemokine gradients cooperatively speed through cell-substratum adhesiveness. Nature 385: 537–540. shape migration patterns of dendritic cells. Immunity 32: 703–713. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021