Cpg oligonucleotides odn 1826

Mouse B cells were isolated by the EasySep Mouse B cell Negative selection Kit (STEMCELL Technologies, Vancouver) according to the manufacturer's protocol. Purity based on CD19 expression was around 95–97% as determined by flow cytometry. Cells were seeded at a cell density of 2.5 x 10⁵ or 3 x 10⁶ mL⁻¹, for flow cytometry staining and ELISA, and RNA extraction, respectively. Cells were stimulated in complete RPMI with 10 μg mL⁻¹ of unconjugated goat anti‐mouse IgM F(ab’)₂ (Jackson ImmunoResearch Laboratories, West Grove, PA), 10 μg mL⁻¹ or 100 ng mL⁻¹ lipopolysaccharide (LPS) from E. coli 0111:B4 (Sigma‐Aldrich), 1 μg mL⁻¹ APRIL (R&D Systems, Minneapolis, MN), 1 μg mL⁻¹ BAFF (R&D Systems), or 1 μg mL⁻¹ CpG oligonucleotides ODN 1826 (InvivoGen, San Diego, CA). To induce IkBα degradation, 1 x 10⁶ cells were stimulated in 100 μL complete RPMI medium for 90 min with 10 μg mL⁻¹ of unconjugated goat anti‐mouse IgM F(ab’)₂ (Jackson ImmunoResearch Laboratories), 50 ng mL⁻¹ PMA and 1 μm ionomycin, or 0.1 μm calyculin A, in the presence of 10 μm cyclohexamide (all from Sigma‐Aldrich).

Mice were immunized intraperitoneally (i.p.) with 50 μg NP40‐Ficoll (Biosearch Technologies, Novato, CA) in 200 μL PBS. CpG oligonucleotides ODN 1826 (InvivoGen) were administered at a dose of 50 μg in 200 μL i.p. LPS of the Salmonella Minnesota strain (Enzo Life Sciences, Farmingdale, NY) was injected intravenously (i.v.) at a dose of 5 μg in 100 μL PBS.