Flow cytometry analysis of peripheral blood

For analysis of the activation/exhaustion/inhibition markers in the NK cells of the peripheral blood, venous blood was collected in EDTA-anticoagulated tubes, and staining was performed using the following antibodies: CD3 (BV605/Clone: SK7), CD19 (Horizon V500/Clone: HIB19), CD16 (APC-Cy7/Clone: 3G8), CD56 (Alexa 700/Clone: B159), CD62L (CF594/Clone: Dreg 56), CD38 (FITC/Clone: HIT2), and PD-1 (PerCP-Cy5.5/Clone: EH12.1) from BD Pharmingen (San Jose, CA, USA) and NKG2A (PE/Clone: 131411; R&D, Minneapolis, MN, USA) and Tim-3 (PE-Cy7/Clone: F38-2E2; Biolegend, San Diego, CA, USA). Approximately 70 µL of whole blood was stained for 20 min and then incubated for 15 min with FACS lysing solution (BD FACS Lysing; BD Biosciences, San Jose, CA) to lyse the erythrocytes. After two washes in an isotonic solution (Hemoton SPEC; Brazil), 500,000 events were acquired using a flow cytometer (LSR Fortessa; BD Biosciences, USA) and were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA).