Mouse splenocytes were harvested for analysis with IFN-γ ELISpot as previously described [43 (link)], using restimulation with 2 µg/mL of a pool of 275 peptides. These peptides are synthesised as 15 mers and overlapping by 10 amino acids, corresponding to MERS-CoV spike protein, and have been previously tested for mouse and human samples [25 (link),30 (link),31 (link)]. Each sample of splenocytes was plated in duplicate wells at three different numbers of cells: 500,000, 250,000, and 125,000 cells per well; thus, each sample was plated in a total of six wells. Cells were incubated with the peptide for 18–20 h at 37 °C, plates were washed, and spots were developed using a Mouse IFN-γ ELISpot kit (MabTech, Nacka Strand, Sweden), following the manufacturer’s instructions. The total number of spots in each well was counted using an AID ELISpot reader (AID Autoimmun Diagnostika, Straßberg, Germany). The numbers of spots were calculated to report the spots per 1 million splenocytes for each well. Further duplicate wells were plated with 250,000 cells from each sample and left without peptide restimulation. In the absence the restimulation, the frequencies of IFN-γ+ cells were subtracted from the cellular frequencies of the tested restimulated samples. IFN-γ secreting splenocytes were reported as the average of spot forming cells (SFCs) per million splenocytes for each sample.
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