Anti cd23

Bone marrow, spleen, mesenteric lymph node (mLN) and Peyer’s Patch (PP) cells were isolated as described previously (26 (link)). In brief, cells were stained with anti-B220 (BioLegend), anti-CD11b (BD Biosciences), anti-CD43 (eBioscience), anti-CD24 (BioLegend), anti-BP1 (eBioscience), anti-IgD (BioLegend), anti-IgM (BioLegend), anti-CD93 (eBioscience) and anti-CD23 (BioLegend) for B cell development staining. For B1 and B2 cell development staining, cells were stained with anti-B220 (BioLegend), anti-CD19 (BioLegend), anti-CD43 (eBioscience), anti-CD23 (BioLegend) and anti-CD5 (BioLegend). For FOB and MZB cell staining, cells were stained with anti-B220 (BioLegend), anti-CD19 (BioLegend), anti-CD21 (BD Biosciences) and anti-CD23 (BioLegend).Cells were stained with anti-B220 (BioLegend), anti-Gl7 (BioLegend), anti-CD95 (Fas) (BD Biosciences), anti-CD86 (BioLegend), and anti-CXCR4(BioLegend) for GC B cell staining and cells were stained with anti-B220 (BioLegend), anti-MHC II (eBioscience) and anti-CD86 (BioLegend) for B cell activation staining.

A full list of antibodies is provided in the Key Resource table (Table 1). The following antibodies were used for FACS analysis: Anti-mouse/human CD45R/B220, anti-CD23, anti-CD21/CD35 (CR2/CR1), anti-CD93 [AA4.1], anti-mouse IgM, anti-IgD, anti-CD45.1, anti-CD45.2, anti-CD3ε, anti-Ly-6G/Ly-6C(Gr-1), anti-CD11b, anti-TCRb, and anti-CD5 were all purchased from Biolegend. Anti-Mouse CD19 was purchased from BD Biosciences. Rabbit Anti-Ki67 (Novocastra), rabbit anti-cleaved caspase 3 (Cell signaling), rabbit anti-Phospho-Histone H3 (Ser10) (Cell signaling), rat anti-Pax5 (Biolegend), Guinea pig anti-Cytokeratin 8+18 antibody, and FITC rat Anti-mouse/human CD45R/B220 (BioLegend) were used for fluorescence immunohistochemistry. Anti-rabbit Alexa Fluor Cy3 (Jackson ImmunoResearch), anti-rabbit Alexa Fluor 647 (Jackson Immunoresearch), anti-rat Cy3 (Jackson Immunoresearch), and Alexa Fluor anti-guinea pig 647 secondary antibodies were used for in fluorescence immunohistochemistry. Notch2 (D76A6) XP (Cell signaling) and HRP-linked rabbit IgG (GE healthcare) secondary antibodies were used for western blot analysis.

Splenocytes (2 × 10⁶ cells/well) were stimulated with 10 μg/mL anti-IgM (31178, Invitrogen) or 10 μg/mL anti-IgD Ab (2057001, Invitrogen) for 4, 14, and/or 24 hours. Splenocytes were stained with fluorescent conjugated anti-CD25 (PC61.5, Tonbo Biosciences), anti-CD69 (H1.2F3, BioLegend), and anti-B220 (RA3.6B2, BioLegend) or anti-B220 (RA3-6B2, BioLegend), anti-CD93 (AA4.1 BioLegend), anti-CD21 (7E9, BioLegend), and anti-CD23 (B3B4, BioLegend), and they were analyzed by flow cytometry.