Cell id iridium intercalator solution

Sample preparation. Cryopreserved PBMC were thawed, resuspended in phosphate buffer saline (PBS) to a concentration of 1-3x10⁶ cells/mL and incubated 10 min with FcR Block Reagent (Miltenyi Biotec) prior to Ab staining.
Staining procedure. Purified antibodies were either purchased pre-conjugated from the manufacturer (Fluidigm, San Francisco, CA) or conjugated in-house with the appropriate metal isotope using MaxPar Metal labeling kits (Fluidigm) according to manufacturer’s instructions. For staining, 1x10⁶ cells were washed in Maxpar cell staining buffer (Fluidigm) and stained in 100 μL Maxpar cell staining buffer (Fluidigm) containing the cocktail of 24 Abs listed in Supplemental Table 1. Stained cells were then incubated for 10 min in 1.6% paraformaldehyde (Sigma). DNA staining was performed by overnight incubation in 2 mL of 125 nM Cell-ID Iridium intercalator solution (Fluidigm) at 4°C. Cells were then washed, pelleted, and kept at 4°C until acquisition.
Data Acquisition. Samples were analyzed on a CyTOF2 mass cytometer upgraded to Helios (Fluidigm). Cells were resuspended in EQ™ Four Element Calibration Beads (Fluidigm) diluted to 0.5X in Maxpar ultra-pure water (Fluidigm) and filtered twice through a 50 µm nylon mesh to reach an acquisition rate of 200-500 events per second.

Extracellular staining was performed on pooled barcoded cells in Maxpar Cell Staining Buffer (Fluidigm) for 30 minutes at room temperature. Intracellular staining (perforin) was performed in Maxpar Perm-S Buffer (Fluidigm) for 30 minutes at room temperature. Stained cells were then incubated for 10 minutes in 1.6% formaldehyde (FA) freshly prepared from 16% stock FA (Sigma-Aldrich). DNA staining was performed by overnight incubation at 4°C in 2mL of 125nM Cell-ID Iridium intercalator solution (Fluidigm). Cells were then washed, pelleted, and kept at 4°C until acquisition the same day.