Elispot mouse ifn γ elispot set

IFNγ ELISpot assays (BD ELISPOT Mouse IFNγ ELISPOT Set, BD Biosciences) were performed according to the manufacturer’s instructions. Briefly, 96-well ELISpot plates were coated with an IFNγ capture antibody (BD Biosciences, 51-2525K) in PBS overnight at 4 °C. Plates were then blocked with complete growth medium for 2 h at room temperature. Wells were then emptied, and 1 × 10⁶ splenocytes from immunized mice were added to the plates. Cells were arrayed in the presence or absence of 15-mer peptides with 11-residue overlaps derived from the SARS-CoV-2 Spike_RBD (10 μg/mL) in 200 μL of complete medium and incubated overnight at 37 °C. Plates were then washed and incubated with a biotinylated IFNγ detection antibody (BD Biosciences, 51-1818KZ) for 2 h at room temperature and incubated with streptavidin-horseradish peroxidase (BD Biosciences) for 1 h at room temperature. Plates were developed with 3-amino-9-ethyl-carbazole substrate (BD ELISPOT AEC Substrate Set) for 5 min to 30 min and dried overnight. Spots were enumerated using the KS ELISpot analysis system (Immunospot).

ELISPOT was performed according to BD Biosciences manufacturer instructions (BD ELISPOT Mouse IFN-γ ELISPOT Set cod. 551083). 5x10⁵ splenocytes or 2 x10⁵ tumor infiltrated lymphocytes (TIL) were counted and plated in each well. In brief, tumor biopsies were cut into small fragments ∼2–3 mm in length and subjected to a commercial mechanical/enzymatic dissociation system (GentleMACS, Miltenyi Biotec, Bergish Gladbach, Germany). After disaggregation, the single cell suspension was passed through 70-μm strainers. TIL were isolated from dissociated tumors by CD45 (TIL) MicroBeads (Miltenyi biotec), for positive selection of CD45-specific TIL. Enriched TILs were cultured in complete media (RPMI 1640 (Lonza) supplemented with 10% fetal calf serum (FCS), 1% glutamine, 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (all from Life Technologies, Paisley, UK).
Both splenocytes and TILs were stimulated with 10ug/ml of single or peptide pool used for the immunization and incubated for 24-26h. As negative and positive control, peptide diluents PBS and 5ug/ml of phorbol myristate acetate (PMA, Sigma-Aldrich) were used respectively. The plates were read with an AID EliSpot Reader Systems (AID GmbH, Strassberg, Germany). The results were calculated as spot forming counts as a mean of a duplicate count from the specific antigen stimulation minus the negative control.

ELISPOT was performed according to BD Biosciences manufacturer instructions(BD ELISPOT Mouse IFN-γ ELISPOT Set cod. 551083). 2.5 × 10⁵ splenocytes were counted and plated in each well in duplicate. Cells were stimulated with 2 ug/ml of Trp2 peptide as well as with 5 ug/ml of phorbol myristate acetate (PMA, Sigma-Aldrich) and incubated for 24–26 h. As negative control PBS was used. The plates were read with an AID EliSpot Reader Systems (AID GmbH, Strassberg, Germany). The results were calculated as spot forming counts as a mean of a duplicate count from the specific antigen stimulation minus the negative control.