Maxpar reagent kits

Cells were thawed for 3 minutes at 37°C in culture medium. 20 U/mL heparin sodium salt and 25 U/mL benzonase nuclease were added to prevent cell clumping (Sigma-Aldrich, St. Louis, MO). CyTOF staining was performed at room temperature. Cells were stained with Cell-ID cisplatin (Fluidigm, San Francisco, CA) for viability, washed, and blocked with TruStain FcX (BioLegend, San Diego, CA) for 10 minutes. Cells were then stained with CyTOF antibodies labeled with rare earth metal isotopes using MaxPar reagent kits from Fluidigm (Table S2, Supplemental Digital Content 1) for 30 minutes. After fixation and permeabilization of the cells using the FoxP3 Staining Buffer Set (eBioscience, San Diego, CA), samples were barcoded using combinations of palladium isotopes as described (18 (link)), allowing the entire mixture of samples to be analyzed in a small number of CyTOF runs over 2 days. 18.75 μM iridium intercalator solution (Fluidigm, San Francisco, CA) was added to the cells, and cells were subsequently washed and reconstituted in Milli-Q filtered distilled water with EQ four element calibration beads (Fluidigm, San Francisco, CA). Cells were analyzed on a Helios CyTOF Mass Cytometer (Fluidigm, San Francisco, CA) (19 (link)).