Maxpar metal conjugated antibodies

Thawed PBMCs (~3×10⁶ 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 60×10⁶ 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 ~5×10⁵ 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.