Igm pe cy7

Lymphocytes were isolated as described and stained with anti-mouse IgD PerCP-Cy5.5 (1:400, 405710), LPAM (integrin α4β7) PE (1:100, 120605; (Biolegend, San Diego, CA), B220 V500 (1:400, 561227), CD19 APC-H7 (1:200, 560143), CD80 PE (1:500, 553769), CD273 APC (1:200, 560086), CD138 PE (1:200, 553714), IgM PE-Cy7 (1:200, 552867; BD Biosciences), CCR9 PE-Cy7 (1:100, 25-1991), CD73 PE-Cy7 (1:50, 25-0731), IgA PE (1:50, 12-4204), GL7 eFluor 450 (1:100, 48-5902), CD38 Alexa700 (1:800, 56-0381), CD21/35 Pacific Blue (1:800, 57-0212; eBiosciences) or CCR10 APC (1:100, FAB2815A; R and D systems. Minneapolis, MN) and were analysed using LSR II (BD Biosciences) or Navios (Beckman Coulter, Brea, CA) flow cytometers. For sorting, cells were labelled with anti-mouse CD138 PE (1:200, 553714), CD19 PE-Cy7 (1:200, 552854), CD80 APC (1:200, 560016) and GL7 FITC (1:100, 553666) before sorting using a FACSAria (BD Biosciences). Cells were sorted into tubes that had been coated with 2% BSA/PBS overnight, and pelleted by centrifugation at 600 g before being resuspended in PBS and injected into recipient mice or used for gene sequence analysis.

CH12 cells were plated at 50,000 cells per ml in complete RPMI supplemented with anti-CD40 antibody (1 μg/ml, Miltenyi), IL-4 (20 ng/ml, Miltenyi), and TGF-β (1 ng/ml, R&D Biotech) to induce IgM to IgA switching. After 3 days, cells were assayed for class switching by flow cytometry using IgA-PE (eBiosciences 12-4204-82, clone mA-6E1, 1:200) and IgM-PE-Cy7 (BD Biosciences 552867, clone R6–60.2, 1:200 dilution) antibodies, and a Fortessa analyser (BD Biosciences). Data were analyzed by FlowJo v10.4.2 software. The gating strategy is provided in Supplementary Fig. 8.