Cpg 2006

Purified naïve B cells were cultured in B-cell medium (RPMI 1640 containing L-glutamine; Invitrogen Life Technologies, CA, USA), supplemented with 10% fetal calf serum (FCS) (Invitrogen Life Technologies, Waltham, MA, USA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES) (pH 7.4; Sigma-Aldrich, St Louis, MO, USA), 0.1 mM nonessential amino acid solution (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen Life Technologies), 60 mg ml⁻¹ penicillin, 100 mg ml⁻¹ streptomycin, 40 mg ml⁻¹ transferrin (Sigma-Aldrich), and 20 μg ml⁻¹ Normocin (InVivogen, San Diego, CA, USA); and stimulated with 100 ng ml⁻¹ CD40L alone (Enzo, Farmingdale, NY, USA) or with IL-4 (100 ng ml⁻¹), IL-21 (50 ng ml⁻¹; both Peprotech), or CpG 2006 (1 μg ml⁻¹, Invitrogen, Carlsbad, CA, USA), APRIL (500 ng ml⁻¹, Adipogen, San Diego, CA, USA), in the presence or absence of IL-4 and IL-21. For some experiments, B cells were labelled with division-tracking dye cell trace violet (CTV, Invitrogen).^{32 (link)} For phenotypic and functional analysis, cells were cultured in 96-well plates for 5 or 6 days, collected, stained with CD20, CD27, CD38, IgG, IgM, IgA and the proportion of isotype switched and differentiated antibody secreting cells determined as previously described.^{6 (link)} Secreted IgM, IgG and IgA levels were determined by Ig Heavy chain-specific immunoassays as previously described.^{6 (link)}

Thiostrepton and azithromycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). TLR ligands, including Pam3Cys, polyinosinic-polycytidylic acid (polyI:C), LPS, and R848, were purchased from InvivoGen (San Diego, CA, USA). CpG-2006 was purchased from Invitrogen (Carlsbad, CA, USA) or Genomics BioSci & Tech (New Taipei City, Taiwan). LL37 was purchased from GeneDireX (Gueishan Township, Taiwan). Human recombinant IFN-γ and IL-4 were purchased from PeproTech (Rocky Hill, NJ, USA).