Flow Cytometric Profiling of Immune Cells

Flow cytometric analysis was performed in accordance to published guidelines (30 (link)). Single cell suspensions of bone marrow (BM), mesenteric lymph nodes (mes-LN) and mediastinal lymph nodes (med-LN) were generated by mechanical disruption. Erythrocytes in the BM were lysed with ACK-buffer (0.15M NH₄Cl, 1mM KHO₃, 0.1mM Na₂EDTA) and all cells resuspended in FACS buffer (PBS, 2% FCS, 1 mg/mL NaN₃). For staining of IgE-expressing cells cytophilic IgE was efficiently removed by short treatment with acetate buffer as described (26 (link)). Fc receptors were blocked with anti-mouse CD16/CD32 mAb (BioXcell) for 5 min at RT and the primary antibodies were incubated for 25 min at 4°C in the dark (for list of antibodies used refer to Supplementary Table S1).
As secondary staining streptavidin conjugated to BUV 395 was added and the cells were incubated for 20 min at 4°C in the dark. For PCs and GC B cells during primary and secondary infection IgA, IgE and IgG1 were instead intracellularly stained using Cytofix/Cytoperm™ (BD Bioscience) and Phosflow™ Perm/Wash Buffer I (BD Bioscience). Cells were analyzed on a BD LSR-Fortessa instrument (BD Bioscience).