Immunospot series 4 analyzer

IFNγ, IL-4, and IL-17 ELISPOT assays were performed as previously described using capture and detecting anti-mouse cytokine antibody from BD Pharmingen (3 (link)–5 (link), 22 (link)). Mouse IL-21 ELISPOT assay kit was purchased from eBioscience and used per manufacturer’s instructions. Recipient spleen cells were stimulated with mitomycin C-treated BALB/c or SJL spleen cells or with 3μM HY_Dby peptide for 24 h. The frequencies of IgG and IgM secreting ASCs were determined using ELIS pot ^PLUS for mouse IgG or IgM kits (MABTECH AB, Nacka Strand, Sweden) as previously published (20 (link)). The resulting spots were analyzed using an ImmunoSpot Series 4 analyzer (Cellular Technology, Cleveland, OH).

ELISPOT assays to enumerate donor-reactive T cells in the spleens of cardiac graft recipients were performed as previously detailed (10 (link), 11 (link)). Briefly, splenic responder cells and mitomycin C-treated T cell depleted autologous or donor stimulator spleen cells were cocultured for 24 h at 37°C in serum-free HL-1 media in 96 well plates coated with anti-IFN-γ or anti-IL-2 capture mAb (BD Biosciences). To compare alloreactive CD4⁺ and CD8⁺ T cell priming, each cell population was purified from spleen cell suspensions using magnetic bead chromatography (Stem Cell Technologies) and then these purified responder cells were stimulated with T-cell depleted splenocytes. After culture, all cells were washed from the plate and biotinylated anti-IFN-γ or anti-IL-2 detecting mAb (BD Biosciences, San Jose, CA, USA) were added, followed by anti-biotin alkaline phosphatase. After development with the chromagen, the total number of spots per well was quantified using an ImmunoSpot Series 4 Analyzer (Cellular Technology Ltd., Shaker Heights, OH).

IFN-ɣ ELISPOT assays were performed as previously described using capture and detection anti-mouse IFN-ɣ antibodies from BD Biosciences (San Diego, CA) (31 (link)). Recipient spleen cells were stimulated with mitomycin C–treated BALB/c or SJL spleen cells for 16–24 h. Spots were developed and analyzed using an ImmunoSpot Series 4 analyzer (Cellular Technology, Shaker Heights, OH).