Protocol development
Well established and tested approaches were adapted for ease and
simplicity31–33. A schematic presentation of the
protocol is depicted in Figure 1.
In brief, blood was collected using EDTA coated
Vacuette® tubes. Peripheral blood mononuclear cells
(PBMCs) were isolated by density gradient centrifugation technique with
BioColl separating solution permitting cell separation based on
polysucrose polymers. Short-term cell culture maintenance was performed
by Roswell Parm Memorial Institute (RPMI) 1640 medium supplemented with
GlutaMAXTM, 1% Penicillin/ Streptomycin, and 10%
fetal bovine serum (FBS). For flow cytometric screening of human
samples, SARS-CoV-2 spike protein was labeled with fluorescein (FITC) or
Cy5 fluorochromes using Lightning-Link® rapid labeling kits. This allows
direct linkage of SARS-CoV-2 specific S- protein by robust covalent
binding and fast processing of the assay as corresponding conjugates are
ready-to-use in less than twenty minutes. Labeling the protein of
interest independently with two different fluorochromes before
incubation of the sample and analyzing the double-positive cells greatly
enhances the specificity31,32. Subsequently, only
double-positive B cells binding S-FITC and S-Cy5 were subjected to
further evaluations. Another control measure to ensure specificity is to
demonstrate that the response can be blocked by excess unlabelled
SARS-CoV-2 S-protein, which blocks the S-protein specific B-cell
receptors, thereby inhibiting the binding of the S-FITC and S-Cy5 to the
B cells33.
A detailed list of all materials and reagents, as well as devices used,
is provided in Table 1.
We designed a staining panel to enable the detection of SARS-CoV-2
specific B cells by applying a set of fluorophore-coupled antigens
characteristic to different B cell subsets such as naïve, switched, and
unswitched BMEMORY cells. Detailed information on the
panel is provided in Table 2.