Automated elispot plate reader

Enumeration of antigen-specific IFN-γ secreting cells was done by ELISpot assay, as described before [3 (link),5 (link),10 (link),25 (link)]. Briefly, spleen cells (4 × 10⁵/well) were added to ELISpot plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), followed by incubation in the presence of appropriate antigen-specific stimulant at a concentration of 2 µg/mL for 20 h at 37 °C, 5% CO₂. A peptide corresponding to CD⁸⁺ T cell epitope OVA_257-264: SIINFEKL (JPT Peptide Technologies GmbH, Berlin, Germany) was used as a stimulant. To measure background responses, cells were incubated in the absence of any stimulants. The plates were developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was added to visualize the spots. Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA).

IFN- γ ELISpot was also conducted as previously described^{10 (link)}. The levels of spike glycoprotein specific T cells were quantified by ELISpot using a mouse IFN-γ kit (Mabtech Inc., Cincinnati, OH, USA). A spike peptide library (JPT Peptide Technologies GmbH) based on the reference strain sequence and consisting of 315 peptides (15mers overlapping by 11 amino acids with last peptide consisting of a 17mer) was used to stimulate splenocytes isolated from each of the mice. The library was split into three subpools and used to separately stimulate 4 × 10⁵ cells in duplicate at a final concentration of 2 µg/mL per peptide. Cells were also incubated without any stimulants to measure background responses. Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA). For each animal, values obtained with media alone were subtracted from those obtained with each of the spike peptide pools, and then combined to yield an overall number of antigen-specific IFN-γ⁺ SFC/10⁶ splenocytes per animal.