Quantifying Mucosal Immune Responses

Mixed cellulose ester membrane-bottom plates (Millipore) were coated with rT2544 or anti-mouse total immunoglobulin overnight at 4 °C. Wells were blocked with 1% BSA and incubated for 2 h at 37 °C. Cells isolated from mesenteric lymph nodes (MLN), Peyer’s Patches (PP), and spleen of immunized mice was added to blocked wells for 5 h, followed by washing with PBST. The plate was incubated with enzyme-conjugated anti-mouse IgG and IgA (Southern Biotech) overnight at 4 °C and developed with substrate. The number of spots was counted separately for each well. In a separate experiment, IFN-γ (ELISpot set from BD Bioscience) and IL-17 (ELISpot kit from R&D) pre coated mixed cellulose ester membrane-bottom plates were incubated with cells isolated from Peyer’s Patches (PP) stimulated with rT2544 for 24 h. The plate was incubated with enzyme-conjugated anti-mouse IFN-γ and IL-17 antibodies and developed with substrate. The number of spots was counted separately for each well.