Multiparametric Flow Cytometry of Lymphocytes

Collection and preparation were previously described (12 (link)). Red blood cells were lysed using ice-cold deionized water for 30 s. White blood cells were then resuspended in FACs buffer [1× phosphate buffered saline (PBS), 2% heat-inactivated fetal bovine serum (FBS)], counted, and blocked for 10 min in FACS Buffer and Human Trustain FcX Fc receptor blocking solution (Biolegend; #422302) at room temperature, followed by staining with CD3-APC/Fire750 (BioLegend; #344840), CD56-PE (BioLegend; #318306), CD8-PerCP (BioLegend; #344708), CD11a-PE/Cy7 (BioLegend; #301220), CD16-FITC (BioLegend; #302006), CD69-APC (BioLegend; #310910). The corresponding immunoglobulin G (IgG) isotype controls were used for staining the lymphocytes. Cells were analyzed on a BD FACSCanto™ II, and the FACs data were analyzed using FlowJo V.10 data analysis software.