To detect antibody binding to SARS‐CoV‐2‐S protein, HEK‐293T cells were cotransfected with SARS‐CoV‐2‐S DNA and a GFP reporter plasmid (e.g., pEGFP‐C1) using the PEI method as described previously [63 (link)]. A total of 105 thawed or freshly transfected cells were incubated first in 96‐well plate with 100 μL undiluted hybridoma supernatant or 100 μL mouse serum (1:200 dilutions in R10+ medium), bound antibodies were detected with a Cy5‐conjugated goat anti‐pan‐mouse IgG antibody (Southern Biotechnology, Birmingham, USA, #SBA‐1030‐15). Cells were washed in FACS buffer and analyzed with an Attune Nxt, CytoFlex, or a Gallios flow cytometer (Thermo Fisher Scientific or Beckman Coulter, Brea, USA, respectively) and evaluated with Flow LogicTM (IInivai Technologies Mentone, Australia). Flow cytometric analysis adhered to “Guidelines for the use of flow cytometry and cell sorting in immunological studies” [64 (link)].
Hybridoma cells were stained intracellularly for the assessment of the monoclonality of the colonies and the determination of the IgG subtype. Briefly, 105 cells were fixed for 20 min in 2% paraformaldehyde (Morphisto, Frankfurt am Main, Germany) diluted in PBS, washed twice with FACS buffer, resuspended in permeabilization buffer (0.5% Saponin Sigma Aldrich, in FACS buffer) containing fluorochrome‐conjugated murine H and L isotype‐specific antibodies (anti‐mouse IgG1‐APC # 550874 and anti‐mouse IgG3‐bio # 020620 from BD, Franklin Lakes, USA, anti‐mouse IgG2b‐PE SBA‐1090‐09 and anti‐mouse IgG2c‐bio SBA‐1079‐08 from Southern Biotech, anti‐mouse Ig light chain lambda APC # 407306 and anti‐mouse IgG light chain kappa PE both from Biolegend, San Diego, USA) and incubated for 1 h at 4°C. Cells were again washed in FACS buffer and analyzed with an Attune Nxt, CytoFlex, or a Gallios flow and evaluated with Flow LogicTM.
Hybridoma cells were stained intracellularly for the assessment of the monoclonality of the colonies and the determination of the IgG subtype. Briefly, 105 cells were fixed for 20 min in 2% paraformaldehyde (Morphisto, Frankfurt am Main, Germany) diluted in PBS, washed twice with FACS buffer, resuspended in permeabilization buffer (0.5% Saponin Sigma Aldrich, in FACS buffer) containing fluorochrome‐conjugated murine H and L isotype‐specific antibodies (anti‐mouse IgG1‐APC # 550874 and anti‐mouse IgG3‐bio # 020620 from BD, Franklin Lakes, USA, anti‐mouse IgG2b‐PE SBA‐1090‐09 and anti‐mouse IgG2c‐bio SBA‐1079‐08 from Southern Biotech, anti‐mouse Ig light chain lambda APC # 407306 and anti‐mouse IgG light chain kappa PE both from Biolegend, San Diego, USA) and incubated for 1 h at 4°C. Cells were again washed in FACS buffer and analyzed with an Attune Nxt, CytoFlex, or a Gallios flow and evaluated with Flow LogicTM.
