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.