Stimulating Splenic B Cells to Detect Cytokines and Induce Tregs

Mouse splenic CD19⁺ B cells (1.5x10⁶/ml) were cultured in medium (RPMI 1640 glutamax; Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 100 μg/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and restimulated for 2 days with SEA (20 μg/ml) to allow the detection of cytokines, as previously established for in vivo schistosome-exposed B cells [36 (link)]. Supernatants were collected for cytokine analysis by ELISA. Cells were cultured for an additional 4 hours with Brefeldin A (10 μg/ml; MedChemExpress) to detect intracellular IL-10 by flow cytometry. For in vitro Treg cell induction, SEA-stimulated CD19⁺ B cells were co-cultured with MACS-sorted CD4⁺CD25^- T cells at a 1:1 ratio (1 x 10⁶/ml each). After 4 days, the frequencies of Treg cells were determined by flow cytometry by gating on Foxp3⁺CD25⁺ cells in the CD3⁺CD4⁺ T cell population, and culture supernatants were collected for subsequent cytokine determination.