Mouse ifn γ elispot plus hrp kit

IFN-γ-producing splenocytes from vaccinated or naive unvaccinated mice were analyzed using a Mouse IFN-γ ELISpot ^PLUS (HRP) kit (Mabtech). Spleens were minced in RPMI 1640 Medium (Thermo Fisher Scientific) and cells were filtered through a 70-μm sterile cell strainer (Corning). Red blood cells were lysed using red blood cell lysis buffer. Remaining splenocytes were washed and adjusted to a concentration of 1 × 10⁶ cells ml^-1 in complete RPMI 1640 Medium. 100 μl cells/well were added to conditioned ELISpot plates and each recombinant antigen, stSpA, stSbi or stEsxA, was used as the inducer with a final concentration of 2 μg/ml. Plates were incubated at 37°C in a humidified incubator with 5% CO₂ for 24 hours. Following 2 h primary antibody incubation and 1 h secondary antibody incubation at room temperature, spots were developed using TMB substrate solution and recorded in an immunospot analyzer.

Spleens from mice were collected and processed into single-cell suspensions in RPMI1640 media supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin (R10 media). Red blood cells were lysed using eBioscience RBC lysis buffer (Thermo Fisher Scientific) and resuspended in R10 media to stop the lysis. Splenocytes were counted and 100,000 cells per well were added to the plates of the Mouse IFN-γ ELISpot^PLUS (HRP) kit (Mabtech). Cells were then stimulated for 18 hours at 37°C with SARS-CoV-2 S PepTivator peptides (Miltenyi Biotec) or media alone as a negative control. Spots were developed as per the manufacturer’s instructions. Spots were quantified by eye and plotted as SFU per million cells.