Cultured B-1 cells selleck kinase inhibitor were stained with PE-labelled anti-CD138 antibody (clone 281-2) (all antibodies from BD Pharmingen). For assessment of proliferation, freshly isolated B-1 cells were stained with 2 μmol/l CellTrace™ CFSE (Invitrogen), according to the manufacturer’s protocol, before the experiment. At the end of

the experiment, cells were harvested and directly resuspended for analysis. For apoptosis assays, cultured B-1 cells were stained with FITC-labelled annexin V (FITC annexin V apoptosis detection kit; BD Pharmingen) and cell viability solution containing PI3K inhibitor 7-aminoactinomycin D (7-AAD) (BD Pharmingen),

according to the manufacturer’s instructions. Cells were analysed using a FACS Aria II (BD Pharmingen) and at least 100 000 cells were counted per sample in in-vivo experiments and at least 5000 cells in in-vitro experiments, with dead cells excluded based on FSC. Specific IgM and IgG antibodies were determined in plasma and in cell culture supernatants by chemiluminescent ELISA, as described previously [7]. For detection of total IgM, microtitre plates were coated with purified rat anti-mouse IgM (2 mg/l) (clone II/41; BD Pharmingen). For the analysis of specific

IgMs, microtitre plates were coated with copper oxidized (CuOx)-LDL (5 mg/l), MDA-LDL (5 mg/l) or Pneumovax CHIR-99021 solubility dmso (10 mg/l). CuOx-LDL and MDA-LDL were prepared from human LDL, as described previously [25]. Plates were post-coated with Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and samples were incubated for 1 h. Serum samples were diluted in TBS containing 1% BSA to a final dilution of 1:300 for detection of total IgM, 1:100 for IgM against CuOx-LDL and MDA-LDL and 1:50 for IgM against Pneumovax. Cell culture supernatants were diluted 1:125 for detection of total IgM and IgM against CuOx-LDL and MDA-LDL. Antibodies in samples were detected with alkaline phosphatase-conjugated goat anti-mouse IgM (μ-chain specific; Sigma-Aldrich) and quantified with Lumiphos 530 (Lumigen, Inc., Southfield, MI, USA) using LMaxII (Molecular Devices, Sunnyvale, CA, USA). Total RNA from isolated peritoneal B-1 cells and positive control tissue (mouse liver, skeletal muscle and placenta) was extracted with the RNeasy micro prep kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.