Elispot Assay for T cell response
The IFNγ and IL-2 responses of T cells were studied with Fluorospot Assay (Abcam Fluorospot, USA) according to the manufacturer’s instructions. Thus, plates were incubated for 30 seconds at room temperature with 35% ethanol and washed with DPBS 1X. The capture antibodies were added and incubated overnight at +4°C. After washing with DPBS 1X, RPMI containing 10% FBS cell culture media was added into the wells and incubated for 2 hours at room temperature. Plates were washed with DPBS 1X and 100.000 cell suspension was added into each well with appropriate concentration of SARS-CoV-2 S peptide activator (PepTivator®SARS-CoV-2 Prot S-research grade (6nmol/peptide, Miltenyi Biotec, Germany). Cells were incubated at 37°C in a CO2 incubator for 20 hours and were washed 3 times. Detection antibodies were added and incubated for 1 hour 30 minutes at RT. After washing, FITC-green fluorescence conjugate/Streptavidin-phycoerythrin solution (for IFNγ/IL-2 respectively) was added to each well and incubated for 1 hour at RT at dark. Cells were washed three times again and all residuals buffer was removed with distilled water. After drying, spots were read with GFP and RFP filters, Leica M205 FA under a dissection microscope. The PMA/Ionomycin was used as a positive control, DMSO was used as a negative control, CMV virus was used for cross-reaction, and tests were performed in duplicate.