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Single-Cell Mass Cytometry of Differential Immune and Drug

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Single-Cell Mass Cytometry of Differential Immune and Drug RESEARCH ARTICLE Performance assessmentofmasscytometry. The workflow for mass cytometry is comparable with that of fluorescence flow cytometry (Fig. 1A). Single-Cell Mass Cytometry of Differential Antibodies coupled to distinct, stable, transition element isotopes were used to bind target epitopes on and within cells. Cells, with bound antibody- Immune and Drug Responses Across isotope conjugates, were sprayed as single-cell droplets into an inductively coupled argon plasma a Human Hematopoietic Continuum (created by passing argon gas through an induc- tion coil with a high radio-frequency electric cur- 1 1 2 3 1 1 Sean C. Bendall, * Erin F. Simonds, * Peng Qiu, El-ad D. Amir, Peter O. Krutzik, Rachel Finck, rent) at approximately 5500 K. This vaporizes 1,7 3 1 4,5 6 Robert V. Bruggner, Rachel Melamed, Angelica Trejo, Olga I. Ornatsky, Robert S. Balderas, each cell and induces ionization of its atomic con- 2 1 3 4,5 1 Sylvia K. Plevritis, Karen Sachs, Dana Pe’er, Scott D. Tanner, Garry P. Nolan † stituents. The resulting elemental ions were then sampled by a time-of-flight (TOF) mass spectrom- Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used eter and quantified. The signal for each transi- single-cell “mass cytometry” to examine healthy human bone marrow, measuring 34 parameters tion element isotope reporter was integrated as simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The each cell’s constituent ions reached the detector. signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 Currently, TOF sampling resolution enables the simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. measurement of up to 1000 cells per second. The data set allowed for an algorithmically driven assembly of related cell types defined by surface We compared mass cytometry with conventional antigen expression, providing a superimposable map of cell signaling responses in combination with drug nine-parameter fluorescence flow cytometry in inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both analysis of cytokine signaling through responses precise signaling responses that were bounded within conventionally defined cell subsets and in human peripheral blood mononuclear cells more continuous phosphorylation responses that crossed cell population boundaries in unexpected (PBMCs) from two healthy donors (Fig. 1, B to E, manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide and fig. S1). Seven surface antigens (CD3, CD4, on May 5, 2011 system-wide views of immune signaling in healthy human hematopoiesis, against which drug action CD8, CD45RA, CD56, CD20, and CD33) and anddiseasecanbecomparedformechanisticstudies and pharmacologic intervention. two intracellular phosphoprotein epitopes [phos- phorylated signal transducer and activator of luorescence-based flow cytometry has single cells to create a detailed response profile transcription 3 and 5 (pSTAT3 and pSTAT5)] were been fundamental to the discovery and of the healthy primary human hematopoietic sys- measured by means of fluorescence cytometry on Fdefinition of major and minor cell subsets tem with 34 simultaneously measured cellular two human PBMC samples treated with interleukin-2 of the immune system. Although the outline of parameters. This allowed us to take full advantage (IL-2), IL-6, IL-10, granulocyte-monocyte colony hematopoiesis is generally understood (1), a com- of the measurement resolution of mass spectrom- stimulating factor (GM-CSF), or interferon-a prehensive framework of its system-wide proper- etry and apply it to single-cell analysis. Because (IFNa) to measure cytokine-mediated signaling www.sciencemag.org ties remains to be determined (2). Technological the method is largely unhampered by interference responses in specific cell subsets. In traditional developments in flow cytometry and cell sorting from spectral overlap, it allows for the detection flow cytometry, forward scatter (FSC) and side [the introduction of new fluorophores, such as of considerably more simultaneous parameters scatter (SSC) measurements of laser light are used quantum dots (3)] have paralleled appreciation than does traditional flow cytometry (17, 18). to detect the presence of a cell and to “trigger” the of the compartmentalization of function in the Combined with its quantitative nature, atomic mass electronics in order to collate information as a hematopoietic system and contributed to diverse spectrometry measurement creates a platform with cell “event” (the window of time during which a fields, including immunology, stem cells (4, 5), which to conduct multiplexed measurement of cell is measured). Because FSC and SSC are not HIV (6), cancer (7), transcription (8, 9), intra- single-cell biological parameters that can exhibit currently implemented on the CyTOF platform, Downloaded from cellular signaling (10, 11), apoptosis, cell cycle vastly different dynamic ranges during signaling alternative parameters providing analogous utility (12), and development of cytometry-based clin- or over time (such as signaling changes indicated were included to assist with the discrimination of ical diagnostics (13, 14). However, use of flow by shifts in protein phosphorylation). single-cell events: (i) an antibody to the surface cytometry remains practically confined to the We simultaneously measured 34 parameters epitope CD45 (expressed on most cells measured measurement of 6 to 10 simultaneous param- in each single cell in human bone marrow (BM) in this study), (ii) a metal-encoded DNA inter- eters (15). Analysis at the 11- to 15-parameter samples to provide an in-depth analysis of normal calator to identify nucleated cells (20), and (iii) a range is possible but limited by compensation human hematopoietic and immunological signal- derived parameter (“cell length”) indicating the needed to correct for spectral overlap that can ingoverlaidontoadetailed template of cell phe- duration of each cell’s measurement window (18). create a source of confounding variability (16). notype. Cell subset–specific signaling phenotypes Fluorescence (Fig. 1B and fig. S1A) and mass We used transition element isotopes not nor- of drug action in the face of clinically meaningful (Fig. 1C and fig. S1B) cytometry analysis pro- mally found in biological systems as chelated anti- physiologic stimuli were localized to pathway and vided comparable results when analyzed via tra- body tags in atomic mass spectrometric analysis of cell-specific boundaries, with examples in B cell ditional dot plots (fig. S2). Pertinent qualities, + 1 signaling shown. These provide a system-wide such as reduced CD45RA expression on CD4 Baxter Laboratory in Stem Cell Biology, Department of Micro- view of signaling behaviors, expanding our view T cells relative to that on CD8+ cells, were re- biology and Immunology, Stanford University, Stanford, CA 94305, USA. 2Department of Radiology, Stanford University, Stanford, CA of drug action while allowing us to limit the func- produced between platforms (Fig. 1, B and C). 94305, USA. 3Department of Biological Sciences, Columbia Uni- tions that certain drugs might have on complex Despite use of different metrics for identifying versity, New York, NY 10027, USA. 4University of Toronto, Toronto, tissues. Given that this technology can reason- cell events, both platforms yielded quantitatively 5 ON M5S 3H6, Canada. DVS Sciences, Markham, ON L3R 6E7, ably be expected to allow for as many as 100 pa- similar frequencies (P < 0.000001) for 12 man- Canada. 6BD Biosciences, San Diego, CA 95131, USA. 7Biomedical Informatics Program, Stanford University, Stanford, CA 94305, USA. rameters per cell (18, 19), it affords an opportunity ually gated cell populations in the parallel anal- – *These authors contributed equally to this work. to increase our understanding of cell type specific ysis of two separate door samples (fig. S1C and †To whom correspondence can be addressed. E-mail: signaling responses in complex, distributed or- table S1). Patterns of specific induction of STAT [email protected] gans such as the immune system. protein phosphorylation within the CD4+CD45RA+ www.sciencemag.org SCIENCE VOL 332 6 MAY 2011 687 RESEARCH ARTICLE A Nebulize Single-Cell Droplets Cell 1 Antibodies Labeled with Elemental Isotopes ICP-MS Cell 2 Elemental Analysis Mass Cytometer Cell 3 Cytobank.org Mass 2D Plots Expression & SPADE Analysis Upload Fold-Change .FCS Integrate Files Signal Isotope B Isotope A BCFluorescence Cytometry* Mass Cytometry 104 104 104 250K 105 105 CD8 CD4 CD8 CD4 200K T Cells T Cells 103 103 T Cells 103 T Cells 104 104 150K 5 3 102 102 5 102 3 SSC 5 10 103 DNA 100K 103 10 10 10 CD45RA CD45RA CD45RA CD45RA on May 5, 2011 102 50K 102 6 0 4 0 0 6 0 4 0 -102 -102 104 -10 -10 -10 00 0 50K 100K 150K 200K 250K -102 0 102 103 104 105 -102 0 102 103 104 105 0 25 50 75 100 125 -10 0 10 102 103 104 -10 0 10 102 103 104 FSC CD8 CD4 Cell Length CD8 CD4 4 10 4 4 250K 10 10 105 105 1.CD33 7.NKT 2.CD20 3 200K 10 Myeloid 103 2.CD20 103 Cells 1.CD33 B Cells Myeloid 104 104 7.NKT B Cells 150K 2 Cells 10 102 102 CD3 SSC CD3 CD45 CD20 3 100K 10 103 CD20 10 10 10 NK 50K 2 10 102 NK 0 0 0 0 0 Cells www.sciencemag.org -102 -102 8 9 Cells -10 -10 -10 8 9 0 -102 0 102 103 104 105 -102 0 102 103 104 105 -102 0 102 103 104 105 -10 0 10 102 103 104 -10 0 10 102 103 104 -10 0 10 102 103 104 CD33 CD3 CD56 CD33 CD3 CD56 D Unstimulated IL-2 IL-10 IFN α E pSTAT3 pSTAT5 105 Fluorescence Population: 123456789 123456789 Cytometry Unstim Fluorescence 4 10 IL-2 Cytometry 103 IL-6 IL-10 Downloaded from 102 0 GM-CSF -102 α ** -102 0 102 103 104 105 IFN 104 pSTAT5 Unstim Cytometry 3 10 Cytometry IL-2 Mass Mass 102 IL-6 IL-10 10 GM-CSF 0 IFN α -10 -100 10 102 103 104 pSTAT3 -5.1 0 1.5 Fig.
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