Sars cov 2 virus peptivator pool

Mouse Spleen T cells were centrifuged with Phosphate Buffer Saline (PBS) at 300xg for 10 min. Pellet was resuspended in TEXMACS (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) cell culture media (%3 human AB serum and 1% Pen/Strep). 500,000 cells in 100 µl were added into microplate already coated with a monoclonal antibody specific for mouse IFN-γ. Either 3 µg/ 100 µl inactivated SARS-CoV-2 or 1000 nM SARS-CoV-2 virus PEPTIVATOR pool (SARS-CoV-2 S, N, and M protein peptide pool) (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) were added into each well including mouse spleen T cells. The microplate was incubated in a humidified 37 °C CO₂ incubator. After 48–72 h incubation, IFN-γ secreting cells were determined with Mouse IFNγ ELISPOT Kit (RnDSystems, USA) according to the manufacturer’s instructions. The spots were counted under the dissection microscope (Zeiss, Germany).

500,000 cells isolated from mouse spleen were incubated with 1000 nM SARS-CoV-2 virus PEPTIVATOR pool (SARS-CoV-2 S, N, and M protein peptide pool) (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) in a humidified 37 °C CO₂ incubator. After 48–72 h incubation, the mouse cytokine profile was analyzed using the supernatant of the cultures using the MACSPLEX Cytokine 10 kit (Miltenyi Biotec). Also, in order to determine T cell activation and proliferation, the restimulated cells were stained with the antibodies including CD3, CD4, CD8, CD19, and CD25 as an activation marker (Miltenyi Biotec). The Cytokine bead array and the T cell activation and proportions were analyzed using the MACSQUANT Analyzer (Miltenyi Biotec).