Elispot kit

Granzyme B (GrB)-secreting cells were spotted and enumerated using ELISPOT kits and assay protocols obtained from R&D Systems (Minneapolis, MN). Briefly, 96-well PVDF membrane plates were coated overnight at 4°C with an anti-GrB capture mAb. Membranes were subsequently blocked for 2 hours and washed before erythrocyte-depleted splenocytes from T Ag-primed WT and IDO^−/− mice were seeded at 3×10⁵ cells/well and stimulated with indicated peptides. Concanavalin A (ConA) was used as a positive control at a final concentration of 5 µg/mL. The plates were incubated overnight at 37°C and 6% CO₂ in a humidified atmosphere. Cells were then removed and the plates were washed before a biotin-labeled polyclonal Ab detecting mouse GrB was added to the wells. After overnight incubation at 4°C, the plates were washed and 100 µL of streptavidin-alkaline phosphatase (1∶60 in PBS/1% BSA) was added to each well. The plates were incubated at room temperature for an additional 2 hours and spots were visualized with BCIP/NBT (5-bromo-4-chloro-3′ indolylphosphate p-toluidine salt and nitro blue tetrazolium chloride in organic solvent) and subjected to automated evaluation using an ImmunoSpot S5 UV Analyzer (Cellular Technology Ltd., Cleveland, OH).

Mouse granzyme B was measured by an ELISpot kit (R&D BioSystems, USA) according to the manufacturer’s instruction. In brief, 5 × 10⁴ cells/well were seeded in antibody-coated wells in triplicate and stimulated by 2 µg/ml of E7 (amino acids 49–57) peptide. The plates were incubated for 24 h at 37 °C, 5 % CO₂. Following wash steps and incubation with a biotinylated detection antibody, alkaline-phosphatase conjugated streptavidin was added. Unbound enzyme was subsequently removed by washing and BCIP/NBT substrate solution was added. The blue-black colored precipitates formed at the site of cytokine localization, and appeared as spots, with each individual spot representing an individual mouse granzyme B secreting cell, were counted manually using a stereomicroscope.

HA-specific antibody responses were determined using a standard enzyme-linked immunosorbent assay (ELISA) with avidin-biotin system [32 (link)]. H5N1 A virus A/Vietnam/1203/2004 recombinant HA (BEI Resource) at a concentration of 2 μg/mL was used. Biotinylated goat anti-mouse IgG, IgG1, and IgG2 (1 : 5000 dilution) (R&D Systems, Minneapolis, MN), 1.25 mM pNPP phosphatase substrate (MP Biomedicals, Santa Ana, CA), and 2.21 mM hydrogen peroxide (Millipore, Temecula, CA) were used.
IFN-γ and IL-4 secreting cells were determined using an ELISpot kit (R&D Systems). Goat anti-mouse IFN-γ and IL-4 antibodies were used (Millipore, USA). Splenocytes (1 × 10⁶ cells/well) that were freshly prepared from vaccinated mice were seeded to the plates and stimulated with HA-specific peptides (ISVGTSTLNQRLVP) (BEI Resources). This peptide was derived from A/Vietnam/1203/2004 H5N1 HA. The plates were treated sequentially with biotinylated goat anti-mouse IFN-γ (1 : 5000 dilution) and IL-4 antibodies (1 : 5000 dilution), alkaline phosphatase conjugated streptavidin (1 : 1000 dilution), and the substrate solution (BCIP/NBT Chromogen) to reveal the spots. The spots were analyzed using ELISpot (CTL ImmunoSpot Analyzer, OH, USA).

The numbers of PRRSV-specific interferon-γ-producing cells (IFN-γ-PC) were determined using ELISPOT kit (R&D Systems, Minneapolis, MN, USA). Briefly, 2 × 10⁵ PBMC were stimulated with either homologous or heterologous PRRSV at 0.01 MOI or PHA (10 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 20 hours at 37 °C in 5% CO₂. Spots were counted by an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH, Strassberg, Germany). PRRSV-specific IFN-γ-PC was expressed as spot forming colonies per million of PBMCs in each well.