Mass Cytometry: A new platform for single cell characterization Marie Iannone Biological Sciences Research Triangle Park, NC

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1 Mass Cytometry: A new platform for single cell characterization Marie Iannone Biological Sciences Research Triangle Park, NC ELRIG, Ware

2 How does mass cytometry progress state of the art single cell analysis? Massive Multiparametric Analysis Fusion of two well-established technologies: flow cytometry and inductively-coupled plasma mass spectrometry. Number of parameters possible by conventional flow cytometry is limited by spectral space and spill-over. By switching from fluorochrome labels to mass tags we can expand our parametric scope from ~15 to 50 per cell. Chattopadahay et al. Immunology Volume 125, ELGIG, Ware UK

3 Utilizing the Power of the Atomic Spectrum Each vertical bar represents a stable isotope that can be measured by mass cytometry

4 Mass Cytometry How it works Bendall et al. TREIMM, Volume 33, 2012

5 Flow Cytometry vs. Mass Cytometry Comparison of utility and performance Flow Cytometry Mass Cytometry Measurement basis Fluorescent probes Stable mass isotope probes Maximum number of parameters 6-18 ~35 Sensitivity range (arbitrary units) Signal crosstalk Compensation Careful panel design Sampling efficiency >95% <30% Measured cells/sec 25, Reagent cost per probe/test $2.00-$8.00 $1.50-$3.00 Adapted from Bendall et al. TREIMM 33(7): (2012).

6 How is it being used? Bendall & Simonds et al., Science 332, 687 (2011) Bone marrow cells 13 surface markers 18 additional intracellular markers to probe signaling pathways (phosphorylation of kinase substrates)

7 Making sense of complex staining patterns by aligning Like near Like SPADE (spanning-tree progression analysis of density-normalized events) Qiu et al., Nature Biotechnology, 2011

8 Use of 13 surface markers to define multiple hematopoietic subpopulations SPADE Cell-Type Specific Signaling Bendall & Simonds et al., Science 332, 687 (2011)

9 How is mass cytometry being used? Recent High-Profile Publications: Profiling of kinase signaling and/or cytokines in complex cell populations (immune cells/ bone marrow). Mass tag cellular barcoding for compound screening. Imaging.

10 Sweating the small stuff Panel design Sources of Mass Tag Crosstalk 1. Impurities (0-4%) 2. Oxides (0-3%) Not all metals have 100% purity Mass plus 16 channel 3. Abundance Sensitivity (0-1%) Mass +/- 1 channels

11 Sweating the small stuff Panel design Mass Minus One Mass Plus One 161Dy Oxidation of 145Nd

12 Sweating the small stuff Panel design MaxPar Panel Design Tool 26 custom conjugations 6 custom conjugations

13 Sweating the small stuff Antibody coupling, QC and validation of binding Maleimide-based coupling chemistry. Each in-house coupled antibody requires: QC for mass tag abundance (atoms of mass tag per antibody molecule). Validation that antibody bind its intended target. Antibody titration to determine optimal binding concentration. CD279 Expressing Jurkat Line Dilution Dilution 3 Dilution Dilution 4 Dilution No Antibody 0 Parental Jurkat Line Dilution 2 0 No Antibody 0

14 Sweating the small stuff Strategies to improve sensitivity For low abundance antigen expression: Avoid mass channels with cross talk from other labels Use mass tags in the Dalton range Employ labeling strategies using mass tag-labeled secondary antibodies (i.e. anti-fitc, anti-biotin). Covalently modify primary amines with a sulfhydryl groups, enabling maleimide-based conjugation. Incubate with multiple clones of monoclonal antibody that recognize different epitopes on the same target with all antibodies coupled to the same mass tag. Mass Cytometry Anti-FITC 175Lu Alone Flow Cytometry Anti-FOXP3 FITC + Anti-FITC 175Lu For low abundance targets: optimize signal, minimize noise

15 Cytokine Staining 30 Parameter Panel PMA Ionomycin Unstimulated CD4+ PMA Ionomycin Unstimulated CD8+ TNFa IFNg IL-21 MIP1b IL-2 IL-17A

16 Cytokine Staining SPADE analysis Unstimulated Stimulated IFNg IL-2 Unstimulated Stimulated Unstimulated TNFa IL-21 Colored by cytokine staining intensity; node size by cell count Stimulated MIP1b IL-17A

17 Acknowledgements Biological Sciences (RTP): Tom Consler Florence Perrin Ken Pearce Kendra Hightower Eddie Wood Dave Morris DVS Sciences (Fluidigm): Scott Tanner Clare Rogers Anita Kant Michelle Poulin Jeannie Gaylor Tempero (Cambridge): Jonathan Hill Mike Nolan Christine Miller Scott Davis HIV DPU (RTP): David Favre Richard Dunham Kevin Brown ipts: Juliet McComas Tom Meek John Baldoni

18 Cytokine Staining 30 Parameter Panel Gating Strategy CD4+ Unstimulated 4-Hr PMA Ionomycin Stimulated