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.