Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators
- Experiment Overview
- Figure 1A: Mass-tag cell barcoding
- Figure 1B: Mass-tag cell barcoding
- Figure 1C: Mass-tag cell barcoding
- Figure 1D: Mass-tag cell barcoding
- Figure 2A: PBMC signaling time-course experiment
- Figures 2B-C: PBMC signaling time-course experiment
- Figure 2D: PBMC signaling time-course experiment
- Figure 2E: PBMC signaling time-course experiment
- Figure 3A: Signaling response comparison of PBMCs from eight donors
- Figure 3BCDF: Signaling response comparison of PBMCs from eight donors
- Figure 3E: Signaling response comparison of PBMCs from eight donors
- Figure 4A-B: Analysis of PBMC response to kinase inhibition
- Figure 4C: Analysis of PBMC response to kinase inhibition
- Figure 4D: Analysis of PBMC response to kinase inhibition
- Figure 5: Overview of inhibitor impact
- Figure 6A-D: Principle component analysis of cell type and drug response
- Figure 6E: Principle component analysis of cell type and drug response
- Figure 6F: Principle component analysis of cell type and drug response
Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on human samples at single-cell resolution, but instruments process only one sample at a time. Here we describe mass-tag cellular barcoding (MCB), which increases mass cytometry throughput by using n metal ion tags to multiplex up to 2n samples. We used seven tags to multiplex an entire 96-well plate, and applied MCB to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics and cell-to-cell communication, signaling variability between PBMCs from eight human donors, and the effects of 27 inhibitors on this system. For each inhibitor, we measured 14 phosphorylation sites in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional, systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors and revealed off-target effects. High-content, high-throughput screening with MCB should be useful for drug discovery, preclinical testing and mechanistic investigation of human disease.
The goal of this study was to develop a cell multiplexing method to increase the sample throughput of mass cytometry, and to apply this method to human peripheral blood mononuclear cells (PBMCs) for the study of cell signaling dynamics, variability between PBMC donors, and high-content kinase inhibitor profiling.
Samples were gated into the following cell-types
IgM+ B cells, IgM- B cells, CD4+ T cells, CD8+ T cells, CD14-HLA-DR- Monocytes, CD14- HLA-DRmid Monoctyes, CD14- HLA-DRhigh Monocytes, CD14+ HLA-DR- Monocytes, CD14+ HLA-DRmid Monoctyes, CD14+ HLA-DRhigh Monocytes, CD14- Surface-, CD14+ Surface-, NK cells, Dendritic cells
Samples were stimulated with one of the following conditions:
Orthovanadate, IL-2, IL-3, G-CSF, GM-CSF, BCR/FcR-XL, IFN-g, IFN-a, LPS, PMA/Ionomycin
Samples were treated with one of the following inhibitors
AKT-1/2, Sorafenib, BTK Inhib. III, Crassin, Dasatinib, GDC-0941, Go-6983, H89, IKK Inhib., Imatinib, JAK(pan) Inhib., JAK2 Inhib., JAK3 Inhib., Lck Inhib., Lestaurtinib, PP2, Rapamycin, Ruxolitinib, SB-202190, SP600125, Staurosporine, Streptonigrin, Sunitinib, Syk Inhib. IV, Tofacitinib, U0126, VX680
Surface and intracellular markers measured
CD3, CD45, pNFkB, pP38, CD4, CD20, CD33, pSTAT5, CD123, pAKT, pSTAT1, pSHP2, pZAP70, pSTAT3, CD14, pSLP76, pBTK, pPLCg2, pERK, pLAT, IgM, pS6, HLA-DR, CD7
DVS Sciences, Inc. CyTOF™ Mass Cytometer
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