Cd38 bv786 hit2

Flow cytometric assays were performed as described previously (11 (link), 12 (link)). Briefly, PBMCs were thawed and co-cultured in a 24-well plate at 1.0 × 10⁶ cells/well in RPMI 1,640 with 10% fetal calf serum for 24 h at 37°C and 5% CO₂. After overnight culture, the cells were labeled with the following anti-human monoclonal antibodies: CD3-APC Cy7 (SK7; BD Bioscience), CD4-Alexa700 (RPA-T4; BD Bioscience), CD8-BV480 (PRA-T8, BD Bioscience), CD19-BV421 (HIB19, Bio-Legend), HLA-DR-BV605 (G46-6, BD Bioscience), CD38-BV786 (HIT2, BD Bioscience), PD1-phycoerythrin (PE) (EH12.1, Bio-Legend), CD25-PECy7 (BC96; Bio-Legend), and IL7Ra (CD127)-BV650 (A019D5; Bio-Legend). Isotype controls, 7-AAD, and Fc blocker were included in all experiments. Multicolor flow cytometric analysis was performed using a BD FACS Fortessa analyzer (BD Biosciences) and FlowJo ver. 10 software (Tree Star Inc., Ashland, OR).

Fluorescent SARS-CoV-2-specific RBD probes were prepared by combining biotinylated proteins with fluorescently labeled streptavidin (SA). RBD probes were prepared at a ratio of 4 M of trimer to 1 M of SA. Two RBD probes, one labeled with phycoerythrin (PE) and one labeled with Alexa Fluor (AF)-647, were used in this panel to increase the specificity of detection of SARS-CoV-2-specific B cells. Cryopreserved PBMCs were thawed at 37 °C and stained with a flow cytometry panel consisting of viability dye (FVS440UV), IgM FITC, CD3 PE-CF594 (UCHT1), CD27 PE-Cy7 (M-T271), IgG APC-H7 (G18-145), CD138 BV421 (MI15), CD19 BV510 (SJ25C1), IgD BV650 (IA6-2), and CD38 BV786 (HIT2) (all from BD Biosciences). Cells were first stained with a cocktail of RBD probes for 60 min at 4 °C, washed with PBS, stained with the remaining antibody panel, and incubated for 15 min at room temperature. The cells were then resuspended in 2% FBS/PBS after fixing with Cytofix (BD Biosciences) for 30 min at 4 °C. All flow data were acquired on a BD FACSFortessa and analyzed using FlowJo software (BD Biosciences).