Cd24 apc efluor 780

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.

For all staining, cells were stained in PBS with 2% FCS for 20 min at 4°C. BD Aria II was used for flow sorting and BD LSRII was used to collect data. For sorted cells, debris and dead cells were excluded using FSC-SSC. Doublets were excluded using both FSC and SSC singlet gating, then CD19^+ve B cells isolated. For Figures 1C and 2A, CD4 and CD3 stains were also included to exclude contaminating T cells. Antibodies used were specific for and labeled with CD21-FITC, CD3-PE TxR, CD4-PE, IgM-APC, GM-CSF-PE, IL-10-PE, IL-17-alexa fluor647, IFN-γ-FITC (BD Biosciences); CD11b-BV570, CD11b-PE Cy5, CD19-PE, CD19-BV605, CD1d-PerCP Cy5.5, CD21/35-APC, CD23-PE, CD23-alexa fluor 647, CD23-PE Cy7, CD3-FITC, CD3 PE/Dazzle 594, CD38-PE, CD4-PE, CD4-BV605, CD40-PE Cy7, CD43-FITC, CD43-APC, CD5-PE Cy5, CD80-PerCP Cy5.5, CD86-BV421, TIM1-PE, TNFα-BV605 (Biolegend); CD21/35-APC efluor780, CD24-APC efluor780, CD25-efluor450, CD9-APC, DO11.10 TCR-Biotin, IgD-efluor450, MHCII-PE Cy5, CD19-eFluor450, F4/80-APC, IFN-γ-PE Cy7, streptavidin-PE (eBioscience); IgM-TxR, IgM-alexa fluor647 (Southern Biotech); IgM-alexa488, cell tracker green (Molecular Probes), and CFSE (fluka). All analysis was performed using FlowJo Software.