Peripheral (inguinal, axillary, and cervical) lymph nodes, spleen, MLNs, and PPs in GF or AF mice were collected, and single-cell suspension was prepared. To enrich plasmablasts, lymphocytes were depleted by using biotinylated anti-B220 and anti-CD90.2 antibody and streptavidin-conjugated magnetic beads (BD Biosciences). Enriched cells (0.1 × 106 to 0.5 × 106) were added on an ELISPOT plate previously coated with anti-mouse IgE (RME-1, BioLegend). After 8 hours of incubation at 37°C, the plate was washed with PBS containing 2% Tween 20 and incubated with biotinylated monoclonal anti-mouse IgE (JKS-6, BioLegend) followed by streptavidin-conjugated alkaline phosphatase (BD Biosciences). Spots for IgE-producing cells were detected by addition of the bromochloroindolyl phosphate–nitro blue tetrazolium (BCIP-NBT) substrate (Sigma-Aldrich) solution.

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