198pt monoisotopic cisplatin

Each of the disaggregated tumour cell pellet was resuspended in 300 μL of ice-cold PBS, vortexed well and 300 μL of 1 μM 198Pt monoisotopic cisplatin (Fluidigm, 201198) in PBS was added, and incubated for 1 min at RT. The staining was quenched with 20 mL of CSM-E (Cell Staining Buffer—Extracellular) consisting of 5 mg/ml BSA (Sigma Aldrich, A3294), 0.5% v/v FBS (Thermo Fisher, 10270106) and 0.2 mg/mL DNAse1 in PBS. The cells were resuspended and counted. Totally, 3 × 10⁶ cells from each tumour sample were aliquoted into individual 5 mL polypropylene FACS tubes, washed with CSM-E and pelleted. Twenty microlitre of 100 U/mL heparin sodium salt (Sigma Aldrich, H3393) solution in PBS and 1 μL Fc block (BD Biosciences, 558636) was added. The cell mixtures were gently mixed and incubated on ice for 5 min.

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.