Mouse IFNγ ELISPOT assay was performed according to the manufacturer instructions (R&D Systems) with minor modifications. Briefly, 005 GSCs (1 × 105) in 10% RPMI were irradiated (35 Gy) after overnight culture. Splenocytes (1–2 × 106/well) harvested from treated mice (as in (Cheema et al., 2013 (link))) were co-cultured with 1 × 105 irradiated (35Gy) 005 GSCs or FCS-differentiated 005 cells in 12 well plate for 48 hr at 37°C. Stimulated splenocytes (1–2 × 10 5) were plated onto 96-well PVDF-backed microplates coated with monoclonal antibody specific for mouse IFNγ (Mouse IFN-gamma ELISpot kit; R&D Systems). After 24 hr of incubation at 37°C, plates were washed three times, incubated with detection antibody concentrate overnight at 4°C, washed and incubated with streptavidin-AP for 2 hr at room temperature. After washing, the signal was developed with BCIP/NBT Chromogen (R&D Systems) for one hour at room temperature. Spots were identified and counted on an AID Version 3.1.1 ELISPOT reader.
Mouse ifnγ elispot assay
Manufactured by R&D Systems
The Mouse IFNγ ELISPOT assay is a lab equipment product that measures the secretion of interferon-gamma (IFNγ) by mouse cells. It provides a quantitative analysis of antigen-specific T-cell responses.
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2 protocols using mouse ifnγ elispot assay
1
Mouse IFNγ ELISPOT Assay Protocol
2
Mouse IFN-γ ELISpot Assay
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Hydrophobic 96-well plates with 0.45 µm pore size PVDF membranes (EMD Millipore S2EM004M99) were coated with capture antibody according to the manufacturer’s instructions for the Mouse IFN-γ ELISpot assay (R&D Systems SEL485). The stimulating antigen was 1 µg/mL PfCSP (3D7) overlapping 15-mer peptide pool. The positive control for cell stimulation was 1 µg/mL hamster anti-mouse CD3e (BD Biosciences 553057). The negative control was culture media alone. Plates were blocked with complete media for at least 2 h. Mouse splenocytes were plated based on spot counting optimization and ranged from 25,000 to 100,000 cells/well. Plates were incubated for 42 h at 37 °C with 5% carbon dioxide for cell stimulation. Wells were probed with the detection antibody according to the manufacturer’s instructions. Following the detection incubation, plates were developed with the ELISpot Blue Color Module according to the manufacturer’s instructions (R&D Systems SEL002) and allowed to dry completely before analysis. Spot counting was performed using an AID ELISpot Reader (Autoimmune Diagnostika).
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