Foxp3 fix and permeabilization kit

Serum, spleen, and tumor tissue samples were collected from BP mice on day 6 after treatment. Twenty-five microliters of serum from each mouse was assayed using the MILLIPLEX mouse cytokine/chemokine panels I, II, and III according to the manufacturer’s protocol (EMD Millipore). The concentration of each cytokine/chemokine present in the serum samples was measured using a Luminex 200 system (Luminex Corporation). Fresh tumor tissues were incubated with RPMI medium containing 1 mg/ml collagenase, 100 μg/ml hyaluronidase (Sigma-Aldrich) at 37°C for 60 min, and manually dissociated to generate single cell suspensions. Single cell suspensions from tumor or spleen tissues were then washed twice with staining buffer and incubated with a cocktail of antibodies targeting surface markers at 4°C for 30 min. Cells were then fixed and permeabilized using the Foxp3 fix and permeabilization kit according to manufacturer’s protocol (eBioscience), and then incubated with a cocktail of antibodies against intracellular markers. Stained samples were analyzed with a FACSCanto II or a Helios mass cytometer (Fluidigm). Antibody details for the flow cytometry and mass cytometry staining panels used in this study are provided in Supplementary Table 1.

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).