Anti mouse ifn γ

Harvested spleens from I.M. vaccinated mice were processed into single cell suspensions with a gentleMACS™ Octo Dissociator and Spleen Dissociation Kit (Miltenyi Biotec). Splenocytes were centrifuged (500 x g, 5 min) and resuspended in complete RPMI medium at a concentration of 10 million cells per mL. Cells were plated in a U-bottom 96-well plate (2 million cells per well) and left to culture overnight at 37°C in 5% CO₂. Cells were centrifuged and resuspended in fresh RPMI complete medium containing 40 μL/mL of PeptTivator® SARS-CoV-2 Prot_S (Miltenyi Biotec, #130-126-700), 1 μL/mL monensin (BioLegend), and 5 μg/mL Brefeldin A (BioLegend). After 6 h of incubation, splenocytes were stained for 30 min at RT with Zombie Green™ Fixable Viability Kit (BioLegend) and blocked with anti-mouse CD16/32 (BioLegend). Following blocking, cell surfaces were stained for 30 min at 4°C with anti-mouse CD8a (Biolegend, PE/Cy5), CD4 (Biolegend, APC), CD3 (BD, BUV395), CD44 (Biolegend, BV711). Surface-stained cells were fixed and permeabilized for 30 min at 4°C with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Then cells were stained with anti-mouse IFN-γ (Biolegend, PE), Granzyme B (Biolegend, BV421), IL-4 (Biolegend, PE/Dazzle 594) and TNF-α (Biolegend, PE/Cy7). Cell fluorescence was quantified using a BD FACSymphony™ A5 Cell Analyzer.