Elispot plus mouse ifn γ

The NALT cells pooled from 8 mice were cultured for 3 d in microplates with feeder splenocytes treated with 50 μg/mL mitomycin for 30 min, in the presence of the IVV antigen. The cell proliferation was examined with alamarBlue (Life Technologies Co.) and represented the difference (Δ) of fluorescence intensity between cases with and without IVV. The culture supernatant was recovered at 16 h and the cytokine-producing cells were examined using the ELISpot PLUS mouse IL-4 and the ELISpot Plus mouse IFN-γ (Mabtech AB, Nacka Strand, Sweden) kits. The results represented the difference of the number (Δ) of cytokine-positive cells/10⁶ cells between cases with and without IVV stimulation.

Spleens from immunized animals were removed at Day 51 for the mice and at Day 110 for the hamsters and splenocytes were isolated for protein S-specific T-cell detection with the ELISpotPLUS mouse IFN-γ or ELISpotPLUS hamster IFN-γ kits (Mabtech) according to the manufacturer’s instructions. Briefly, 3–5 × 10⁵ splenocytes/well and 4 μg/well SARS-CoV-2 spike protein B.1.617.2 (Nanjing Vazyme Biotech Co., Ltd) were mixed and incubated at 37 °C. After 48 h followed by 5 washes with PBS, the probe antibodies (anti mouse IFN-γ, Mabtech, 3321-4APW-2; anti hamster IFN-γ, Mabtech, 3102-4APW-2) were added at a concentration of 1 μg/μl and incubated at room temperature for 2 h. After washing 5 times with PBS, alkaline phosphatase labelled streptavidin was added at a dilution of 1 in 1000 and incubated for 1 h at room temperature. After washing 5 times with PBS, alkaline phosphatase labelled streptavidin was added at a dilution of 1 in 1000 and incubated for 1 h at room temperature. After washing 5 times with PBS, the chromogenic solution (BCIP/NBT-plus) was added and incubated at room temperature for 10 min. Colour development was then stopped with deionized water. The number of dots in the wells of the ELISpot plate was analyzed using an ELISpot reader system (AID ELISpot Reader Classic and AID ELISpot Reader Software, Autoimmun Diagnostika GmbH).