Cd23 pe

Sex matched mice at the age of 6–8 weeks were sacrificed by CO₂ asphyxiation. Peritoneal cavity cells were collected by rinsing with FACS buffer (1x PBS plus 2% fetal bovine serum). Cells from the bone marrow and spleen were lysed with a red blood cell lysis buffer to remove red blood cells and filtered through a 70 μM cell strainer (BD Biosciences, Franklin Lakes, NJ). After washing with cold 1x PBS, cells were counted by trypan blue staining. One million cells were resuspended in 100 μL FACS buffer and labeled with 1 μL antibodies of B220-APC, CD43-FITC, BP-1-PE, CD24-PerCP/Cy5.5, CD19-APC-Cy7, IgM-PE, IgD-PerCP/Cy5.5, IgM-PErCP/Cy5.5, CD93-APC, CD23-PE, CD21-FITC, CD5-PerCP (BioLegend, San Diego, CA). Cell subpopulations were analyzed on BD LSR II violet at The University of Iowa flow cytometry facility.

Freshly isolated splenocytes were stained with H2A-biotin, 7AAD (eBioscience), polyclonal goat anti-mouse IgM Fab-FITC (Jackson ImmunoResearch), CD23-PE (Clone B3B4; BioLegend), B220-PeCy7 (Clone RA3-62B; BioLegend), and CD24 APC-eFluor 780 (Clone M1/69; eBioscience) in DMEM + 2% FBS for 15 min at room temperature. Cells were then washed and stained with anti-biotin Fab APC (gift from J. Cambier, University of Colorado) to visualize H2A-reactive cells without crosslinking the BCR. Cells were washed and resuspended in DMEM + 2% FBS with 1 μmol/L Indo 1-AM (eBioscience), then incubated for 1 h at room temperature. Cells were washed and resuspended at a concentration of 10 × 10⁶ cells/ml in warmed DMEM + 2% FBS and placed on the BD LSRFortessa X-20 Cytometer to record 30 s of baseline readings before stimulation with 10 μg/ml of polyclonal F(ab′)2 goat anti-mouse IgM (Jackson ImmunoResearch). For H2A stimulated controls, H2A was directly conjugated to Dylight 650 using the Dylight 650 Pierce labeling kit (Thermo Fisher Scientific), and cells were directly stimulated with 10 μg/ml of H2A-Dylight 650. Kinetic analyses were performed using FlowJo 8 software.

To isolate splenic follicular or marginal zone B cells by FACS, cell suspensions were stained as described for flow cytometric analysis in PBS- 2% FCS. Cells were stained in 15 mL reaction tubes. The solutions’ volumes were adjusted according to the used cell numbers. Cells were stained with CD19 Brilliant Violet 421 (BioLegend), CD23 PE (BioLegend), CD21 biotinylated (eBiosciene) and Streptavidin Cy5 (ImmunoResearch) and isolated with a purity >99% with the MoFlo cell sorter (Beckman Coulter). In addition, splenic naive B cells were isolated by magnetic cell sorting using the “EasySep™ Mouse B Cell Isolation Kit” from STEMCELL according to the manufacturer’s protocol. Isolated splenic B cells were cultured in complete RPMI1640 with 10% FCS and 10 µg/mL LPS with a density of 2x10⁵ cells/mL (37°C, 5% CO₂).