Cell id rhodium solution

CyTOF staining and analysis were performed as described^{41 (link)}. The antibodies used in the CyTOF analyses are summarized in Supplementary Table 2. Cells were subjected to staining after washing with PBS supplemented with 2% fetal calf serum (FCS, Biosera, Orange, CA, USA) (washing solution). The cells were incubated in 5 μM of Cell-ID rhodium solution (Fluidigm, South San Francisco, CA) in PBS, washed using washing solution, and stained with a mixture of surface-staining antibodies (1:100 dilution). After washing, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The fixed and permeabilized cells were stained with the intracellular antibodies (1:50 dilution). After washing twice, the cells were allowed to rest overnight in 125 nM MaxPar Intercalator-Ir (Fluidigm) diluted in PBS solution with 2% paraformaldehyde at 4°C. The cells were then washed once with washing solution and twice with MaxPar water (Fluidigm) and distilled water with minimal heavy element contamination to reduce the background level. The cells were resuspended in MaxPar water supplemented with 10% EQ Four Element Calibration Beads (Fluidigm) and then were applied to the Helios instrument (Fluidigm), and data were acquired at a speed below 300 events/second.

CyTOF staining and analysis were performed as described^{20 (link)}. The antibodies used in CyTOF analyses are summarized in Table S3. The cells were subjected to staining after washing with PBS supplemented with 2% fetal calf serum (FCS, Biosera, Orange, CA, USA) (washing solution) followed by incubation in 5 μM of Cell-ID rhodium solution (Fluidigm, South San Francisco, CA, USA) in PBS, washed using the washing solution, and stained with a mixture of surface antibodies. After washing, the cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The fixed and permeabilized cells were stained with intracellular antibodies. After washing twice, the cells were incubated overnight in 125 nM MaxPar Intercalator-Ir (Fluidigm) diluted in 2% paraformaldehyde PBS solution at 4 °C. The cells were then washed once with the washing solution and twice with MaxPar water (Fluidigm), distilled water with minimal heavy element contamination, to reduce the background level. The cells were then suspended in MaxPar water supplemented with 10% EQ. Four Element Calibration Beads (Fluidigm) were applied to the Helios instrument (Fluidigm), and data were acquired at speed below 300 events/s.