Immunophenotyping of SARS-CoV-2–specific B cells from human PBMCs using flow cytometry

For the preparation of spike-tetramer solutions, refer to the application protocol Analysis of SARS-CoV-2–specific B cells using recombinant SARS-CoV-2 proteins and to the data sheet of the SARS-CoV-2 Spike B Cell Analysis Kit, anti-human.
For preparation of the staining cocktail from the SARS-CoV-2 Spike B Cell Analysis Kit, anti-human, 2 μL of each fluorochrome-conjugated antibody, 5 μL of 7-AAD Staining Solution, 5 μL of spike‑tetramer-PE, and 10 μL of spike-tetramer-PE-Vio 770 were mixed and filled up to 100 μL with PEB buffer for each test (5×10⁶ B cells or 10⁷ PBMCs).
▲Note: CD19⁺ cells can be enriched from a single-cell suspension of PBMCs using the REAlease CD19 MicroBead Kit, human according to the data sheet. 

1. Cell number was determined.
2. Up to 5×10⁶ B cells (or up to 10⁷ PBMCs) were resuspended in 100 μL of staining cocktail.
3. Cells were resuspended and incubated for 30 minutes in the dark at 2–8 °C.
4. Cells were washed by addition of 1–2 mL of PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
5. Cells were resuspended in 100 µL of PEB buffer before flow cytometric analysis using the MACSQuant Analyzer.