Multiscreen ha plates

IFNγ enzyme-linked immunospot (ELISpot) assay was performed as previously described61 (link). Briefly, multiscreen-HA plates (Millipore, Bedford, MA) were coated with 5 μg ml⁻¹ anti-hIFNγ-mAb 1-D1K (Mabtech). T cells were seeded in RPMI medium and added at indicated numbers to 1 × 10⁴ tumour cells per well. After 20–24 h incubation at 37 °C in 5% CO₂, a biotinylated secondary anti-hIFNγ antibody (1 μg ml⁻¹, clone 7-B6-1, Mabtech) was added and spots were developed by sequential addition of 1:1,000 diluted ExtrAvidin alkaline phosphatase and BCIP/NBT Liquid Substrate System (Sigma-Aldrich). Spot numbers were determined with the AID EliSpot reader (AID Diagnostika).

Multiscreen HA plates (Millipore-Sigma) were coated with 10 µg/mL NP20-BSA for 4 h before the addition of cells. Plates were then incubated for 14–18 h at 37 °C 10% CO₂. NP-specific antibody was detected with anti-IgG1-HRP and visualized using 3-amino-9-ethylCarbazole (Sigma-Aldrich).

We have published the B16-OVA tumor model previously [57 (link)]. In brief, B16-OVA was a gift from Dr. Economou, University of California at Los Angeles and cultured in RPMI-1640 media (Mediatech, Hernden, VA, USA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 10,000 IU penicillin, 10,000 μg/mL streptomycin, 25 μg/mL amphotericin (Mediatech), 0.05 mM 2-mercaptoethanol (Sigma), and kept under 0.4 mg/mL G418 selection (Research Products International Corp., Mt. Prospect, IL, USA). A vaccine of B16-OVA cells was made by lethally irradiating them (25 Gy) and injecting 10⁷ cells, or saline, i.p. into Nrf2–/– or WT mice. Seven days later, spleens were harvested and OVA-specific IL-4 and IFN-γ producing T cells enumerated by ELISPOT assay by plating 2 × 10⁵/well for 48 h in anti-IL4- or anti-IFNγ-coated (BD Pharmingen, Franklin Lakes, NJ, USA) MultiScreen-HA plates (Millipore Corp, Billerica, MA, USA). Cells secreting IFNγ or IL-4 were detected using the corresponding biotinylated anti-IL-4 or anti-IFNγ antibodies (BD Pharmingen) and 200× diluted horseradish peroxidase avidin D (Vector Laboratories, Burlingame, CA, USA), with spots being developed in the presence of 0.4 mg/mL 3-amino-9-ethyla-carbazole substrate (AEC, Sigma) and counted using an ImmunoSpot Image Analyzer (Cellular Technology Ltd., Cleveland, OH, USA).