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.