Aid elispot reader system version 3

To assess tumor-specific IFN-γ-producing immune cell populations, IFN-γ ELISPOT assays were performed. Six days following last Ad injection, spleens were harvested aseptically from mice, and unicellular splenocytes prepared as previously described [19 (link)]. Prepared spleen cells (2.5 × 10⁵) were co-cultured with pre-irradiated 4T1 (5.0 × 10³ cells; 5 Gy) for 24 hr in the presence of recombinant mouse IL-2 (100 units/mL). IFN-γ ELISPOT assays were performed according to the manufacturer's specifications for IFN-γ ELISPOT kits (BD Biosciences). Colored spots were quantitatively analyzed by a computer-based immunospot system (AID Elispot Reader System, Version 3.4, Autoimmun Diagnostika GmbH, Strassberg, Germany).

After 3 days following the final treatment with PBS, HY-oAd, 9-ING-41, and HY-oAd plus 9-ING-41, spleens were collected aseptically from tumor-bearing mice, and unicellular splenocytes were prepared as described previously (57 (link)). Briefly, the splenocytes were co-cultured with irradiated MB49 (6,000 rad) tumor cells for 18 h in the presence of recombinant mouse IL-2 (100 U/mL; R&D Systems). An IFN-γ ELISpot assay (BD Biosciences) was then carried out as described previously (57 (link)). The spots were measured using a computer-based immunospot system (AID ELISpot Reader System version 3.4; Autoimmun Diagnostika GmbH, Strassberg, Germany).

At 7 days following treatment with PBS, RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF, spleens were obtained aseptically from tumor-bearing mice, and unicellular splenocytes were prepared as described previously [8 (link)]. Prepared spleen cells were co-cultured with irradiated B16-F10 (5,000 rad) tumor cells for 3 days in the presence of recombinant mouse IL-2 (100 U/mL; R&D Systems). An IFN-γ ELISpot assay was then carried out as described previously [8 (link)]. Spots were measured using a computer-based immunospot system (AID ELISpot Reader System version 3.4; Autoimmun Diagnostika GmbH, Germany).