For IgA and pIgR ELISA analysis, fresh feces were collected from individual mice, homogenized in sterile PBS, and centrifuged to remove bacteria and insoluble debris. Fecal samples were diluted in sterile PBS as a 2-fold serial dilution and added to 96-well plates pre-coated overnight (at 4 °C) with 1 µg/ml anti-mouse IgA (Southern Biotechnology) or 0.5 µg/ml anti-mouse pIgR (R&D Systems). Samples were incubated at room temperature for 2 h. For IgA analysis, HRP conjugated anti-IgA (Southern Biotechnology) was added for 1 h. For pIgR ELISA analysis, biotinylated mouse pIgR antibody was added for 1 h, followed by incubation with avidin-HRP for 30 min. Plates were developed using dTMB substrate according to the manufacturer’s instructions (Thermo Fisher Scientific) and analyzed at 450nm using a microplate reader.
For ELISA analysis of cytokines (IL-23p19, IL-1β and IL-6), equal numbers of wild-type and Map3k14-cKO BMDCs were stimulated with CpG or Pam3CSK4 for 48 h, and supernatants were collected for ELISA analysis using an ELISA kits (Thermo Fisher Scientific) according to the manufacturer’s instructions. Detection limits were 16 pg/ml for IL-23p19, 1.2 pg/ml for IL-1β, and 4 pg/ml for IL-6.
For ELISA analysis of cytokines (IL-23p19, IL-1β and IL-6), equal numbers of wild-type and Map3k14-cKO BMDCs were stimulated with CpG or Pam3CSK4 for 48 h, and supernatants were collected for ELISA analysis using an ELISA kits (Thermo Fisher Scientific) according to the manufacturer’s instructions. Detection limits were 16 pg/ml for IL-23p19, 1.2 pg/ml for IL-1β, and 4 pg/ml for IL-6.