Four element calibration beads

Samples were processed as previously described (14 (link)). Formaldehyde-fixed cell samples were incubated with a cell surface antibody cocktail for 1 h at room temperature, washed once with PBS containing 0.5% BSA, permeabilized with methanol on ice for 15 min, washed twice with PBS containing 0.5% BSA, and then incubated with metal-conjugated antibodies against intracellular molecules for 1 h. Cells were washed twice with PBS containing 0.5% BSA and then incubated at room temperature for 20 min with an iridium-containing DNA intercalator (Fluidigm) in PBS containing 2% paraformaldehyde. After intercalation/fixation, the cell samples were washed once with PBS containing 0.5% BSA and twice with water before measurement on a CyTOF mass cytometer (Fluidigm). Normalization for detector sensitivity was performed by using Four Element Calibration Beads (Fluidigm). After measurement and normalization, the individual files were analyzed by first gating out doublets, debris, and dead cell based on cell length, DNA content, and cisplatin staining. tSNE maps and FlowSOM were generated with software tools available at https://www.beckman.com/flow-cytometry/software. org/ by considering all included markers.

Thawed PBMCs (∼3x10⁶ cells) were spun (300 g, 5 min) and resuspended in calcium magnesium-free phosphate buffered saline (PBS). 1μM Cisplatin (Fluidigm) was added for viability staining for 5 minutes before quenching the reaction with MaxPar Cell Staining Buffer (CSB, Fluidigm). After centrifugation (300 g, 5 min), cells were resuspended in CSB at a concentration of 60x10⁶ cells/mL and incubated (RT,10 min) with Fc receptor binding inhibitor before adding 26 MaxPar metal-conjugated antibodies (Fluidigm) against immunophenotypic markers for an additional 30-minute incubation at RT. Stained cells were then washed two times before resuspension in MaxPar fix and perm buffer with 125μM 191/193Ir intercalator for either an hour at RT or 4°C overnight. Cells were then washed twice with CSB and two times with Nuclease-Free water (Thermo Fisher Scientific) followed by filtering through 40μM strainers to remove aggregates. Cells were then counted and resuspended in Nuclease-Free water at ∼5x10⁵ cells/mL with 1:10 volume of four-element calibration beads (Fluidigm) and analyzed on a Helios instrument (Fluidigm) for 250,000 events for each donor at the NIEHS Flow Cytometry Center. Following the manufacturer’s instructions, downstream processing involved normalization by the calibration beads and fcs files were uploaded to Cytobank.