Foxp3 transcription factor fix perm buffer

Metal-labeled Abs were obtained from Fluidigm or labeled using the MaxPar X8 labeling kit (Fluidigm) according to manufacturer’s instructions (see Table S1). Freshly isolated mouse (BSA- or Dynabead-enriched) splenocytes or thawed human mononuclear cells from blood and spleen were stained with 1 mL of 0.25 μM cisplatin (Fluidigm) for 5 minutes at room temperature to exclude dead cells. Cells were then washed with CyFACS buffer (2 mM EDTA, 1% BSA, 1% in PBS) and stained with heavy-metal-labeled Ab cocktail for 30 minutes on ice. Cells were washed twice with CyFACS then fixed with FoxP3 Transcription Factor Fix/Perm Buffer (Thermo Fisher Scientific) for 2 hours. Human surface CyTOF Abs that were sensitive to FoxP3 buffer in our hands (i.e., CX3CR1, CD123, CD33, CD135, CD172a and CD163), were instead stained after fixation and permeabilization for 30 minutes at 4°C. After staining, samples were washed and incubated with 2% paraformaldehyde (Electron) in PBS containing 125 nM Iridium intercalator (Fluidigm) overnight. Cells were washed with water, filtered, and acquired in a CyTOF2 (Fluidigm) at the Stanford Shared FACS Facility.

Cell suspensions from spleen, lung, and skin were incubated with supernatant against CD16/CD32 (clone 2.4G2, produced in house) to block non-specific binding for 15 minutes at 4°C. Cell suspensions were incubated in Ab mixes in mouse FACS buffer (2 mM EDTA, 2% FBS in PBS) for 20 minutes at 4°C. For transcription factor staining, cells were stained with surface Abs and LIVE/DEAD Fixable Blue (Thermo Fisher Scientific) in PBS for 20 minutes at 4°C, then fixed with FoxP3 Transcription Factor Fix/Perm Buffer (Thermo Fisher Scientific) for 2 hours to overnight and stained intracellularly for 30 minutes in 1X Permeabilization Buffer (Thermo Fisher Scientific).