Bp1 pe

Bone marrow, peripheral blood, and splenic B cells from cCD79 and wild type mice were analyzed using the following antibodies: hCD79A-PE (R&D Systems, FAB69201P); hCD79B-PE (abcam, ab33295); mCD79B-AF488 ([HM79], made in house); hCD79A-AF647 ([Curly-14], made in house); B220-BV786 (BD, 563894); B220-APC (BD, 103212); B220-PE (BD, 561878); CD43 ACP-Cy7 (BD, 562866); BP1-PE (BD, 553735); CD24-APC (BioLegend, 101814); IgD-FITC (BioLegend, 405704); IgM-BUV396 (BD, 564025); CD19-FITC (BioLegend, 152404); CD93-APC (BioLegend, 136510); CD23-FITC (BD, 561772); CD1d-PE (BD, 553846); CD95-PE (BD, 554258); GL7-FITC (BD, 553666); fab anti-mIgG (H+L)-AF647 (Jackson ImmunoResearch, 115–606-072); phospho-Syk (Y352)-PE (BD, 557881); PTEN-PE (BD, 560002); mouse IgG1 isotype control-PE (BD, 559320).

After a mouse was euthanized, bone marrow cells were flushed from the femur with RPMI medium supplemented with 10% fetal calf serum (HyClone), penicillin, and streptomycin. Single-cell suspensions of this bone marrow and a portion of the spleen were prepared by passing samples through a 100-μm cell strainer (BD Pharmingen). Red blood cells were lysed, and the remaining cells were stained with monoclonal antibodies conjugated to phycoerythrin (PE), fluorescein isothiocyanate (FITC), or allophycocyanin (APC). The following monoclonal antibodies were used: B220-APC, CD19-PE, CD43-FITC, BP1-PE, CD25-PE, immunoglobulin (Ig)M-FITC, Gr1-PE, Mac1-APC, c-Kit-APC, Sca1-FITC, CD3-PE, and Ter119-APC (BD Pharmingen). Data were collected using FACSCalibur or LSRII flow cytometers (BD Biosciences), and analysis was performed using FlowJo (TreeStar). The sternum and remaining portion of the spleen were fixed in 10% neutral-buffered formalin and embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin (HE) or used for IHC analysis after being heated for 1 h at 60°C and then deparaffinized and rehydrated. IHC for c-Kit/CD117, CD43, TdT, PAX5, B220, IgM, CD3, myeloperoxidase (MPO), and kappa light chain (KLC) was performed as previously described^{45 (link)} and detailed in Supplemental Table 2.