Biotinylated polyclonal goat anti ifn γ ab

The ELISPOT assay was performed as described previously [35 (link)]. Single-cell suspensions were prepared from the draining lymph node (DLN) of subcutaneous tumor-bearing mice treated with GalCer. A total of 2.0 × 10⁵ cells per well were stimulated for 14-16 h with 0, 0.1, or 1 μg/ml of ova SIINFEKL peptide in 96 well MultiScreen filter plates (Millipore, Billerica, MA) previously coated with monoclonal rat anti-IFN-γ antibody (Ab) (clone; R4-6A2) (BD Biosciences, San Jose, CA). The plates were washed and treated with biotinylated polyclonal goat anti-IFN-γ Ab (R&D Systems, Minneapolis, MN) followed by streptavidin alkaline phosphatase. Staining was visualized thorough the addition of a 5-bromo-4-chloro-3-indolyl phosphatase solution (Sigma-Aldrich, St. Louis, MO) and counted manually under 40× magnification. A single-blind reviewer counted the number of cytokine-secreting cells, and all data were generated by analyzing three separate wells per sample.

Single cell suspensions were prepared from whole spleen, tumor-infiltrating leukocytes or tumor draining lymph nodes and 1.5 - 3.0 × 10⁵ cells/well stimulated for 12 hr with the class-I restricted CT26-derived AH-1 peptide (1 ug/ml) in 96 well Immulon II plates (Millipore, Billerica, MA) coated with anti-IFN Ab (R4-6A2) (BD Biosciences). The plates were washed and treated with biotinylated polyclonal goat anti-IFNγ Ab (R & D systems, MN) followed by streptavidin alkaline phosphatase. Spots were visualized by the addition of a 5-bromo-4-chloro-3-indolyl phosphatase solution (Sigma Aldrich) in low melt agarose (Sigma Aldrich) and counted manually under ×40 magnification. The number of cytokine secreting cells was determined by a single blind reader, and all data was generated by analyzing 12 separate wells per sample.