Mass Cytometry: Streamlined Cell Analysis

Mass cytometry staining and data acquisition were performed as previously described (11 (link)). See Supplemental Table 3 for antibody panel information. In brief, PBMCs were thawed and washed with FACS buffer. Then 4 × 10⁶ or fewer cells per patient were stained with live/dead (1 μM 198PT monoisotopic cisplatin; Fluidigm). Cells were incubated in Cytofix fixation buffer, washed, and barcoded using palladium metal barcodes as per the manufacturer’s instructions (Fluidigm). Cells were incubated with Human TruStain FcX (BioLegend) and stained with an antibody master mix for 30 minutes at room temperature. After washing, cells were fixed with 2.4% formaldehyde in PBS containing 125 nM iridium nucleic acid intercalator (Fluidigm) and kept overnight. Cells were cryopreserved and stored at –80°C until thawing for acquisition. Cells were washed and resuspended at a concentration of 1 × 10⁶ cells/mL in cell acquisition solution with 5% EQ beads (Fluidigm). Acquisition was performed using a Helios mass cytometer (Fluidigm) and a standardized acquisition template. FCS files were bead-normalized and debarcoded using Helios software (Fluidigm). Using FlowJo (BD), debris, dead cells, and doublets were excluded.