Cell id cisplatin 195pt

5x10⁶ peripheral blood mononuclear cells (PBMCs) were stained with Cell-ID Cisplatin-¹⁹⁵Pt (Fluidigm, South San Francisco, CA) to discriminate dead cells. PBMCs were then FcR blocked with Human TruStain FcX (Biolegend, San Diego, CA), stained with the antibody mix as shown in Table S2, fixed with 1.6% paraformaldehyde, and stained with Cell-ID intercalator-Ir. Stained PBMCs were analyzed in a Helios CyTOF 3.0 (Fluidigm) following manufacturer’s instructions at the Cancer and Immunology Core at Vanderbilt University. The data was exported as a Flow Cytometry Standard file (FCS) and normalized using EQ bead standards (Fluidigm) following manufacturer’s protocol.

The procedure for antibody staining and gating strategy was described as following^{70 (link)}. Briefly, single cells from mouse penile tumors were prepared using the Mouse Tumor Dissociation kit (Miltenyl Biotec). All isolated cells were depleted of erythrocytes by hypotonic lysis, and blocked for FcγR using CD16/CD32 antibody (clone 2.4G2, BD Biosciences) and incubated with metal-conjugated antibodies targeting cell surface antigens for 30 minutes at room temperature. Cells were washed and incubated with 5 μM Cell-ID Cisplatin-195Pt (Fluidigm) for 3 min for viability staining. Cells were washed and fixed following the steps of the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Fixed cells were stained with metal-conjugated antibodies targeting intracellular antigens for 1 hour at room temperature. Cells were washed and incubated with Nucleic Acid Intercalator-Ir (Fluidigm) at 4^oC overnight to stain all the nuclei. The samples were analyzed with CyTOF instrument (Fluidigm) in the Flow Cytometry and Cellular Imaging Core Facility at MD Anderson Cancer Center. CyTOF data were analyzed and visualized using Cytobank (http://www.cytobank.org).