Anti mouse ifn γ capture antibody

Upon sacrifice, splenocytes from B16F10-sTAC tumor-bearing mice were analyzed for IFN-γ producing cells by enzyme-linked immunosorbent spot (ELISpot) assay. Multiscreen filtration plates (Millipore, Burlington, MA, USA, MSHAN4550) were coated with 0.5 µg/mL of purified anti-mouse IFN-γ capture antibody (Biolegend, San Diego, CA, USA, 505702) overnight at 4 °C. Single-cell suspensions of splenocytes or tumors were plated at 1 × 10⁶ per well. Splenocytes were stimulated with the SIINFEKL peptide (Anaspec, Fremont, CA, USA, AS-60193-1) at 20 µg/mL. After 16 h of stimulation at 37 °C, the cells were removed by washing and spots were developed with a biotinylated anti-IFN-γ detection antibody (Biolegend, 505804) and streptavidin-horseradish peroxidase conjugate followed by NITRO-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indoylphosphate p-toluidine salt substrate (Sigma, B5655-25TAB). Spot numbers were counted, and data were reported as IFN-γ-spot forming cells per 10⁶ cells.

Upon sacrifice, splenocytes from B16F10-sTAC tumor bearing mice and tumor cell suspensions from CT26.CL25 tumor bearing mice were analyzed for IFN-γ producing cells by enzyme-linked immunosorbent spot (ELISpot) assay. Multiscreen filtration plates (Millipore) were coated with 0.5 μg/mL of purified anti-mouse IFN-γ capture antibody (Biolegend) overnight at 4 °C. Single-cell suspensions of splenocytes or tumors were plated at 1×10⁶ and 5×10⁵ per well respectively. For ELISpot assays with splenocytes from B16F10-sTAC tumor bearing mice, cells were stimulated with the SIINFEKL peptide (Anaspec) at 20 μg/mL. Cells from CT26.CL25 tumors were treated with a known H-2D-restricted β-galactosidase peptide (TPHPARIGL; ChemPep). After 16 hours of stimulation at 37 °C, the cells were removed by washing and spots were developed with a biotinylated anti-IFN-γ detection antibody and streptavidin-horseradish peroxidase conjugate followed by NITRO-blue tetrazolium chloride and 5-bromo-4-chloro-3ʹ-indoylphosphate p-toluidine salt substrate (Sigma). Spot numbers were counted, and data were reported as IFN-γ-spot forming cells per 10⁶ cells.