Frozen PBMC and SVC were thawed and stained with an extracellular primary antibody panel containing the following antibodies from BD Biosciences: BUV496 CD16 (Cat# 612944), BUV563 CD56 (Cat# 565704), BUV661 CD38 (Cat# 612969), BUV737 CD69 (Cat# 612817), BUV805 CD45 (Cat# 612891), BB700 NKG2A (Cat# 747926), APC Vio770 CCR5 (Cat# 557755), V500 CD14 (Cat# 561391), BV510 CD19 (Cat# 562947), BV650 CD98 (Cat# 744505), BV711 Sialyl Lewis x (Cat# 563910), PE-Cy5 CD54 (Cat# 555512). From Biolegend: Biotin NKp46 (Cat# 331906), A700 CD63 (Cat# 353024), BV421 Bcl-2 (Cat# 658709), BV510 CD123 (Cat# 306022), BV750 CD3 (Cat# 344845), BV785 HLA-DR (Cat# 307642), PE CD26 (Cat# 302706), PE-Cy7 NKG2D (Cat# 320812), PE-Cy7 CD162 (Cat# 328816), BB515 CD49e (Cat# 130-110-534, Miltenyi), PE-Cy5.5 KIR2DL1/S1 (Cat# A66898, Beckman Coulter), PE-Cy5.5 KIR2DL2/L3/S2 (Cat# A66900, Beckman Coulter). Staining was performed in FACS buffer (PBS with 2mM EDTA (Cat# AM9260G, Ambion), 2% FCS (Cat# F7524, Sigma). After 20 min incubation at room temperature (RT) and in the dark, the cells were washed twice with FACS buffer and further stained with a secondary antibody panel containing streptavidin (Cat# 624294, BD Biosciences) for 15 min at RT in dark. Cells were washed twice with FACS buffer before adding Fix/perm Buffer (diluted ¼ with eBioscience reagents: Fix/perm diluent (Cat#00.5223.56) and Fix/perm concentrate (Cat#00.5123.43)) and incubated for 45 min RT in dark. Cells were then washed twice with perm/MQ buffer (Perm Buffer 10X (Cat#00.8333.56) diluted 1/10 with MQ water). Further, the cells were stained with an intracellular antibody panel containing the following antibodies from BD Biosciences: BUV395 Ki67 (Cat# 564071), BB755-P Perforin (Cat# 624391, BD Horizon custom reagents), BB790-P Granzyme B (Cat# 624296, BD Horizon custom reagents), and eF660 Eomes (Cat# 50-4877-41, eBioscience), PE-Dazzle594 T-bet (Cat# 644828, Biolegend). Live/dead Fixable Aqua Dead Cell stain kit (Invitrogen, 1:100 dilution) was used to distinguish between dead and live cells. After 30 min incubation, the cells were washed twice with perm/MQ buffer and kept in FACS buffer for immediate flow cytometry analysis. Samples were run on a 29-color Symphony (BD Biosciences) with 405, 488, 561 and 639 lasers using the BD FACSDiva™ software (BD Biosciences). Flow cytometry data generated was analyzed using FlowJo V10 (Treestar, USA).
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