194pt monoisotopic cisplatin

2×10⁶ or fewer cells were incubated with 2% of FBS in PBS with 25 μg/mL of 2.4G2 antibody at 4°C for 10 min prior to surface staining with an antibody cocktail at 4°C for 30 min in a 50 μL volume. Cells were incubated with 2.5 μM 194Pt monoisotopic cisplatin (Fluidigm) at 4°C for 1 min. Cells were then washed twice with FACS buffer and barcoded using palladium metal barcoding reagents according to manufacturer’s protocol (Fluidigm). Subsequently, fixation and permeabilization were performed using the Foxp3 fix and permeabilization kit according to the manufacturer’s protocol (Fluidigm). Cells were then stained with an intracellular stain antibody cocktail (Foxp3, Ki67, granzyme B, T-bet, iNos, Eomes) for 30 min at room temperature. Cells were then washed twice with Foxp3 permeabilization buffer, twice with FACS buffer, and incubated overnight in 1.6% PFA PBS with 100 nM iridium nucleic acid intercalator (Fluidigm). Cells were then washed twice with PBS with 0.5% BSA, filtered, and washed twice with water with 0.1% BSA prior to analysis. Samples were analyzed using a Helios mass cytometer based on the Helios 6.5.358 acquisition software (Fluidigm).

2x10⁶ or fewer cells were incubated with 2% of FBS in PBS with 25 μg/mL of 2.4G2 antibody at 4°C for 10 min prior to surface staining with an antibody cocktail at 4°C for 30 min in a 50 μL volume. Cells were incubated with 2.5 μM 194Pt monoisotopic cisplatin (Fluidigm) at 4°C for 1 min. Cells were then washed twice with FACS buffer and barcoded using palladium metal barcoding reagents according to manufacturer’s protocol (Fluidigm). Subsequently, fixation and permeabilization were performed using the Foxp3 fix and permeabilization kit according to the manufacturer’s protocol (eBioscience). Cells were then stained with an intracellular stain antibody cocktail (Foxp3, Ki67, granzyme B, T-bet, iNos, Eomes) for 30 min at room temperature. Cells were then washed twice with Foxp3 permeabilization buffer, twice with FACS buffer, and incubated overnight in 1.6% PFA PBS with 100 nM iridium nucleic acid intercalator (Fluidigm). Cells were then washed twice with PBS with 0.5% BSA, filtered, and washed twice with water with 0.1% BSA prior to analysis. Samples were analyzed using a Helios mass cytometer based on the Helios 6.5.358 acquisition software (Fluidigm).