B cell isolation kit

Primary mouse B cells isolated from the peritoneal cavity or spleen of C57BL/6J mice were isolated by use of kits purchased from Miltenyi Biotec including B cell Isolation kit (130–090–862), CD19 MicroBeads (130–052–201) and B-1a Cell Isolation Kit (130–097–413). Sorted CD19⁺ or B-1a cells were stimulated with LPS (5µg per ml, Sigma), anti-CD40 Abs (10µg per ml; BioXcell) and anti-IgM Abs (5µg per ml; Jackson ImmonoResearch) for 72 hours as described (15 (link)).

Ramos cells, the human Burkitt’s lymphoma cell line, was purchased from the American Type Culture Collection (Manassas, VA, USA). Primary human B cells were purified from healthy donors using either a RosetteSep Human B Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC, Canada) or a B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).
Blood was obtained from healthy donors after obtaining informed consent according to the Declaration of Helsinki. This was approved by the Institutional Review Board at Seoul National University Hospital. Human monocytes and plasmacytoid dendritic cells (PDCs) were isolated using human monocyte isolation kit II (Miltenyi Biotec) or human plasmacytoid dendritic isolation kit (Miltenyi Biotec). Murine bone marrow-derived macrophages (BMMs) were generated by culturing murine bone marrow cells in the presence of 10 ng/mL M-CSF (Sigma-Aldrich, St Louis, MO, USA) for 7 days.

Mouse embryonic fibroblasts (MEF) prepared from 13.5 dpc embryos and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). CD4⁺, CD8⁺, CD19⁺ and CD11c⁺ cells were purified from the spleen with MACS columns (Miltenyi Biotech). Resting B cells were purified from spleen with B cell isolation kit (Miltenyi Biotech). Bone marrow derived dendritic cells were obtained by culture of bone marrow cells in RPMI1640 supplemented with 10% FCS for 7 days with human Flt3L (50 ng/ml). For transfections, cells were transiently transfected with Effectene (QIAGEN). To generate stable NIH3T3 cell transformants, cells were transfected with pCAG-PDLIM1 or empty vector and selected with G418 (500 mg/ml) for 14 days. For the reporter assay, 293T cells were transfected with the ELΑΜ−1 luciferase construct and expression plasmids encoding p65 and PDLIM1, or with the pGL4-NF-κB luciferase construct and expression plasmids encoding TRAF6, MyD88 or IKKβ and PDLIM1. Total amounts of transfected DNA were kept constant by supplementing with control plasmids. Luciferase activity was measured according to the manufacturer’s protocol in the Dual Luciferase Reporter System (Promega). Alternatively, MEF were transfected with the pGL4-NF-κB luciferase construct together with or without the PDLIM1 vector, then stimulated with LPS or poly (I:C) for 5 hr and luciferase activity was measured.