Measurement of SARS-CoV-2 specific B cells
Briefly, SARS-CoV-2 S1/S2-protein (further referred to as S-protein) (Sino Biological Inc.) was divided into three samples. Sample 1 was left unlabelled for blocking, sample 2 was coupled to fluorescein isothiocyanate (FITC), and sample 3 was linked to Cy5 fluorochrome. Lightning-Link® Rapid Conjugation System (Abcam PLC) was used for labelling according to the manufacturers’ protocol. Labelling was performed with 100 µg of protein diluted to a concentration of 1 µg/µL. After the labelling procedure, the protein integrity was checked by a gel electrophoresis. PBMC were divided into the respective samples (blocked and unblocked samples in concentrations of 1:500 and 1:1000 of fluorochrome-labelled S-protein) with 5x106 PBMC each (Fig. 1). Blocking was performed by using 10 the excess unlabelled protein. After blocking, PBMC were surface stained with fluorochrome-labelled antibodies, as indicated in Table 2. Finally, mixed FITC and Cy5 labelled protein was added in concentrations of 1:500 and 1:1000. Cells were stained for 10 min at 4 °C. After washing with phosphate-buffered saline (PBS), samples were stored at 4 °C until measurement on a Cytoflex flow cytometer. Directly before analysis, the samples were stained with DAPI to differentiate live from dead cells. Further information on applied materials and equipment is provided in Table 1.