Mouse ifnγ elispot kit

Mouse Spleen T cells were centrifuged with Phosphate Buffer Saline (PBS) at 300 × g for 10 min. Pellet was resuspended in TexMACs (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) cell culture media (%3 human AB serum and 1% Pen/Strep). 500,000 cells in 100 µL were added into a microplate already coated with a monoclonal antibody specific for mouse IFN-γ. 1000 nM SARS-CoV-2 virus Peptivator pool (SARS-CoV-2 S, N, and M protein peptide pool) (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) were added into each well including mouse spleen T cells. The microplate was incubated in a humidified 37 °C CO₂ incubator. After 48 h incubation, IFN-γ secreting cells were determined with Mouse IFNγ ELISpot Kit (RnDSystems, USA) according to the manufacturer’s instructions. The spots were counted under the dissection microscope (Zeiss, Germany).

After samples were processed into single cell suspensions, PBMCs or splenocytes were plated into a 96-well round-bottom plate, with each well containing cells from an individual mouse in 200 μl of media. Two and a half million cells were used for splenocytes on day 21. Three million cells were used for the splenocytes on day 35. Two million cells were used for PBMCs. Eight and a half million cells were used for the CD4 and CD8 enriched samples. We stimulated each well with 2 μg/mL of HR2 peptide. After 48 hours of stimulated pre-incubation at 37°C, we collected the cells (290 g, 5 min, 4°C) and resuspended in 100 μl of sterile splenocyte media. The cells were then plated for analysis on ELISpot plate (RND systems, Mouse IFNγ ELISpot kit, #505841). The cells were incubated at 37°C for 36 hours to allow time for epitope processing and MHC presentation and then processed the ELISpot plate according to the manufacturer’s instructions. After the plate was processed and dried overnight, the plate was analyzed via an ELISpot plate reader by Dana Farber Cancer Institute’s Center for Immuno-oncology Translational Immunogenics Laboratory. The fold increase in cells for DoriVac treated groups versus bolus groups was quantified.

Mouse Spleen T cells were centrifuged with Phosphate Buffer Saline (PBS) at 300xg for 10 min. Pellet was resuspended in TEXMACS (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) cell culture media (%3 human AB serum and 1% Pen/Strep). 500,000 cells in 100 µl were added into microplate already coated with a monoclonal antibody specific for mouse IFN-γ. Either 3 µg/ 100 µl inactivated SARS-CoV-2 or 1000 nM SARS-CoV-2 virus PEPTIVATOR pool (SARS-CoV-2 S, N, and M protein peptide pool) (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) were added into each well including mouse spleen T cells. The microplate was incubated in a humidified 37 °C CO₂ incubator. After 48–72 h incubation, IFN-γ secreting cells were determined with Mouse IFNγ ELISPOT Kit (RnDSystems, USA) according to the manufacturer’s instructions. The spots were counted under the dissection microscope (Zeiss, Germany).