Cd21 pe cf594

Cryopreserved PBMCs were thawed and washed twice with 10 ml of fluorescence-activated cell sorting (FACS) buffer (1× PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 μl of PBS containing Zombie UV LIVE/DEAD dye at 1:200 dilution (BioLegend, #423108) and incubated at room temperature for 15 min. After washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD phycoerythrin (PE; Southern Biotech, #2030–09), IgM peridinin chlorophyll protein–Cy5.5 (BioLegend, #314512), CD20 allophycocyanin-H7 (BD Biosciences, #560734), CD27 PE-Cy7 (BioLegend, #302838), CD14 brilliant violet (BV) 650 (BioLegend, #301836), CD16 BV650 (BioLegend, #302042), IgG brilliant UV 395 (BD Biosciences, #564229), CD3 BV650 (BD Biosciences, #563916), CD21 PE-CF594 (BD Biosciences, #563474), Alexa Fluor 488–labeled Beta Spike protein (antibodies-online, #ABIN6963740), Alexa Fluor 647– labeled Omicron Spike protein (Sino Biological, #40589-V08H26), and BV421-l abeled Wuhan Spike protein (Sino Biological, #40589-V27B-B). All antibodies were used as per the manufacturer’s instruction, and the final concentration of each probe was 0.1 μg/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACSAria III. FlowJo software v10 (TreeStar Inc.) was used for data analysis.