Cytokine Enzyme-linked immunospot (ELISpot) assay (BioLegend) was performed to analyze IL-4– or IFN-γ–secreting cells. Briefly, splenocytes or CLN cells (3 × 106 cells/mL, 100 μL/well) were seeded into 96-well filtration plates (MultiScreen-HA, Millipore) that were pretreated with anti-mouse IFN-γ or IL-4 antibodies and then stimulated with H3 (4 μg/mL) for 24 h at 37 °C. After removing cells, plates were incubated with biotin-conjugated IFN-γ or IL-4 detection antibody at 37 °C for 1 h, followed by the addition of horseradish peroxidase (HRP)-streptavidin for another 1 h. True Blue Peroxidase substrate (KPL) was used to develop spots, and spots were recorded with Bioreader-6000-E (BIOSYS).
B cell ELISpot assay was used to evaluate the antigen-specific ASCs. Briefly, 96-well filtration plates were precoated with H3 proteins (50 μL/well, 4 μg/mL) overnight at 4 °C, washed, blocked, and then splenocytes or NALT cells (3 × 106 cells/mL, 100 μL/well) were seeded and incubated overnight at 37 °C. After removing cells, HRP-conjugated anti-mouse IgG or IgA antibodies were added for 1 h at room temperature. True Blue Peroxidase substrate was used to develop spots. Results were recorded with Bioreader-6000-E.
B cell ELISpot assay was used to evaluate the antigen-specific ASCs. Briefly, 96-well filtration plates were precoated with H3 proteins (50 μL/well, 4 μg/mL) overnight at 4 °C, washed, blocked, and then splenocytes or NALT cells (3 × 106 cells/mL, 100 μL/well) were seeded and incubated overnight at 37 °C. After removing cells, HRP-conjugated anti-mouse IgG or IgA antibodies were added for 1 h at room temperature. True Blue Peroxidase substrate was used to develop spots. Results were recorded with Bioreader-6000-E.