Anti mouse ifn γ ab

ELISpot assays to detect IFN-γ were performed as previously described (39 (link)). In brief, 96-well filter plates (Millipore) were coated with anti-mouse IFN-γ Ab overnight at 4°C (eBioscience, clone AN-18). Wells were washed and blocked with complete media. Splenocytes were freshly isolated as above and plated at 1 × 10⁶ cells per well with or without 10μM human insulin (Sigma-Aldrich) in complete RPMI 1640 [10% FBS (HyClone), non-essential amino acids, HEPES, sodium pyruvate, penicillin/streptomycin, L-Glutamine, 2 × 10⁵ M 2-ME (Invitrogen)]. Cells were cultured for 72 hours at 37°C in 5% CO₂. After incubation, plates were washed and incubated with biotinylated anti-mouse IFN-γ Ab (eBioscience, clone R4–6A2). Wells were washed and incubated with horseradish peroxidase (HRP)-conjugated streptavidin (BD Biosciences). After washing, 3-Amino-9-ethylcarbazole (AEC) substrate solution (BD Biosciences) was added and wells were monitored for spot development for 4–10 min. Cold, deionized water was used to stop substrate reaction, and plates were dried overnight. Spots were counted using an ImmunoSpot plate reader (Cellular Technology Limited).