Iridium 191 193 intercalator

A live cell barcoding methodology was applied to decrease inter-sample staining variability, sample handling time, and antibody consumption. The barcoded and combined samples were stained with 0.5 μmol/L viability dyes (cisplatin-195pt; 201064; Fluidigm), vortexed for 2 min at room temperature (RT), and then the reaction was terminated using Maxpar Cell Staining Buffer on a rotating shaker (400 rcf) at RT. The cells were then washed and fixed in 1.6% paraformaldehyde in PBS for 10 min at RT on a rotary shaker (500 rpm). The cells were resuspended in pre-cooled Maxpar Cell Staining Buffer to slow the fixation reaction, followed by washing twice with PBS/bovine serum albumin and once with double-distilled water. Finally, the cells were resuspended in 400 μL surface antibody mixture and incubated at 37 °C for 30 min on a rotating shaker (500 rpm) for surface staining. The samples then stored in freshly diluted 2% formaldehyde in PBS containing 0.125 nmol/L iridium 191/193 intercalator (Fluidigm, 201192) at 4 °C overnight.

Cell isolation and staining for CyTOF was performed as described previously (McKechnie et al., 2020 (link); Vendrame et al., 2020 (link)). Briefly, PBMCs were thawed in complete media and counted, then 1 million PBMCs for each participant were kept on ice for ligand panel staining. The remaining PBMCs were used for NK cell purification by negative selection using a human NK Cell Isolation Kit (Miltenyi). PBMCs and isolated NK cells were stained using the viability marker cisplatin (Enzo Life Sciences), as previously described (Fienberg et al., 2012 (link)). They were then barcoded with a two-of-four CD45-palladium barcoding scheme generated in-house, using ¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁶Pd, and ¹⁰⁸Pd. Barcoded samples were pooled and stained with surface antibodies before fixation with 2% paraformaldehyde and permeabilization (eBioscience Permeabilization Buffer). Samples were then stained with intracellular antibodies and incubated in iridium-191/193 intercalator (Fluidigm) for up to a week. Before the analysis by CyTOF, samples were washed and diluted in EQ Four Element Calibration Beads (Fluidigm). Samples were then acquired on a Helios mass cytometer (Fluidigm). These data are available on ImmPort (https://www.immport.org) under study accession SDY1844.