Anti-IFN-γ pre-coated plates (Mabtech) were first blocked with 10% FBS. Then, 2.5 × 105 mouse splenocytes and 1 µg mL−1 SARS-CoV-2 peptide pools (15-mers overlapping by 11; JPT Peptides) were added and incubated overnight for 24 h at 37 °C and 5% CO2. After incubation, biotinylated cytokine-specific detection antibodies (Mabtech), streptavidin-enzyme conjugate, and substrate (Mabtech) were added. Finally, each well was observed under an optical microscope (Zeiss, Germany) and the number of secreting cells was calculated 18 (link),30 (link).
To detect the level of IL-6, a high binding ELISA plate (Biomat, Italy) was separately coated with anti-mouse IL-6 (Southern Biotech) and then diluted samples from supernatant of activated mouse splenocytes were separately added. After 1 h incubation at 37 °C, plates were washed with PBS and then 100 μL of secondary antibody, including anti-mouse IL-6-HRP (Southern Biotech), was added. Then, 50 μL of TMB (Sigma-Aldrich) was added. After 15 min, 100 μL sulfuric acid (Sigma) was added and OD of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and then the serum level of IL-6 was quantified by standard curve 18 (link).
To detect the level of IL-6, a high binding ELISA plate (Biomat, Italy) was separately coated with anti-mouse IL-6 (Southern Biotech) and then diluted samples from supernatant of activated mouse splenocytes were separately added. After 1 h incubation at 37 °C, plates were washed with PBS and then 100 μL of secondary antibody, including anti-mouse IL-6-HRP (Southern Biotech), was added. Then, 50 μL of TMB (Sigma-Aldrich) was added. After 15 min, 100 μL sulfuric acid (Sigma) was added and OD of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and then the serum level of IL-6 was quantified by standard curve 18 (link).
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