Mononuclear cells were isolated from colonic lamina propria and Peyer’s patches, as previously described.25 (link) Briefly, epithelium was dissociated by using 0.5 mM EDTA and the tissues were further treated with 1.25 mg/mL of collagenase at 37 °C.25 (link) To collect mononuclear cells from mesenteric lymph nodes and spleen, the tissues were mashed mechanically and filtered through 70 μm mesh. Lymphocyte separation medium (MP Biomedicals, Santa Ana, CA, USA) was used to isolate peripheral blood mononuclear cells (PBMCs).
For flow cytometric analysis, cells were incubated with 5 µg/mL of an anti-CD16/32 antibody (Fc block, BD Pharmingen, San Diego, CA) for 5 min and stained for 30 min at 4 °C with fluorescence-labeled antibodies (Abs) specific for c-kit (2B8), CD25 (3C7), CD45 (30F-11), CD63 (5A9),11 (link) RORγt (Q31–378) (BD Pharmingen), and FcεRIα (MAR-1) and Foxp3 (FJK-16s) (eBioscience, San Diego, CA). CD4 (RM4–5), CD11b (M1/70), CD11c (N418), CD39 (Duha59), CD45 (30F11), CD49b (DX5), CD73 (TY/11.8), CXCR5 (L138D7), PD-1 (RMP1–30), GATA3 (16E10A23), and Tim-4 (F31-5G3) were purchased from BioLegend (San Diego, CA). Cells were analyzed by using FACSCalibur, FACSCanto II, and FACSAria III flow cytometry systems (Becton Dickinson, San Jose, CA, USA). The full gating strategies for MCs is shown in Supplementary Fig. 10.
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