Sars cov 2 s peptides pool

In order to quantify the percentages of antigen-specific T cells, approximately 1 million splenocytes were seeded into each well and stimulated with SARS-CoV-2 S peptides pool, hCoV-OC43 S peptides pool and hCoV-229E S peptides pool (GenScript), respectively. Cells were co-stimulated with 2 μg/mL anti-CD28 (Biolegend) at 37°C with 5% CO₂ for 1 h. Cells were then incubated with 5 μg/mL brefeldin A (Topscience), 2 μM monensin (Topscience). DMSO was used as negative control. PMA/ionomycin was used as positive control. After a total of 6 h, the LIVE/DEAD Fixable Viability Dyes (Thermo Scientific) was used to exclude dead cells for analysis. Subsequently, cells were performed with a fixation/permeabilization kit (BD Biosciences). The following cytokine antibodies were used: anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-IL-2 (JES6-5H4), and anti-IL-4 (11B11). The percentages of cytokine-specific splenocytes were analyzed by flow cytometry.

Indicated cells were cultured with SARS-CoV-2 S peptides pool (GenScript) at a concentration of 2 μg/well for 18-24 h. Antigen specific cells of monkeys, BALB/c mice and human PBMCs were detected by Monkey IFN-γ ELISPOT kit, Monkey IL-4 ELISPOT kit, mouse IFN-γ ELISPOT kit, mouse IL-4 ELISPOT kit and Human IFN-γ ELISPOT kit (Dakewe) according to the manufacturer’s protocol. Antigen-specific spots were then counted using an S6 ultra immunoscan reader (Cellular Technology Ltd.), and the number of IFN-γ- or IL-4-positive T cells was calculated by ImmunoSpot 5.1.34 software (Cellular Technology Ltd.). The number of spots was converted into the number of spots per million cells and plotted as mean ± SEM.