CyTOF Profiling of NK Cell Activation

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.