Recombinant baff

Bone marrow (BM) cells from OVA sensitized and exposed mice were harvested and introduced in a mouse Pre-B Colony Forming Cell (CFC) assay using methylcellulose complete media for Pre-B Cells (HSC009; R&D Systems, Minneapolis, USA), according to the manufacturer’s instructions. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semi-solid media in response to cytokine stimulation. Cells were cultured in the presence of complete media (containing IL-7) for 3 days. At day 4 recombinant BAFF (50 ng/ml, R&D Systems), BAFF-R-Ig fusion protein blocking BAFFR (100 ng/ml, R&D Systems) or its control protein was added to the cells. Colony Forming Units (CFUs) were counted at day 7.

Splenic B cells were isolated with MACS beads by negative selection according to the manufacturer’s protocol (STEMCELL Technologies). The purity was routinely >96% as assessed by staining with anti-B220. For the in vitro proliferation assay, splenic B cells were labeled with 1 mM CFSE (Sigma-Aldrich) for 10 min in phosphate-buffered saline (PBS) and washed twice with RPMI 1640 medium containing 10% fetal bovine serum (FBS) before plating. B cells at a density of 1 × 10⁶ cells/ml were then cultured in normal culture medium and stimulated with F(ab’)₂ goat anti-mouse IgM (10 µg/ml; Jackson ImmunoResearch), LPS from Escherichia coli O111:B4 (2 µg/ml; Sigma-Aldrich), recombinant BAFF (100 ng/ml; R&D Systems) and anti-CD40 (1 µg/ml; BD Biosciences). Normal culture medium for B cells was 1640 medium with 10% (vol/vol) FBS, 0.05 mM β-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin. CFSE dilution was analyzed by flow cytometry after 72 h of stimulation. In some experiments, 20 μM DFO or 100 μM FAC was applied to the cell culture medium. For the [³H]thymidine incorporation assay, purified B cells were also plated at a density of 2 × 10⁵ cells per well (200 µl in 96-well plates) with various levels of stimulation and cultured for 72 h with the addition of 0.4 mCi [³H]thymidine for the final 12 h of culture.

Splenic B cells were isolated from 10- to 12-week-old MRL/Lpr mice using B Cell Isolation Kit (Miltenyi Biotec). Isolated cells were washed three times with PBS, after which their purity was assessed by flow cytometry (purity was greater than 97%). For B-cell stimulation and survival, 1 × 10⁶ B cells were cultured in RPMI media containing 2 to 10% fetal bovine serum or 10% of anti-CD138 beads pretreated or IgG-coated beads pretreated MRL/Lpr mice serum. Next, cells were stimulated with 500 ng/ml of recombinant APRIL (Peprotech) or recombinant BAFF (R&D Systems) with or without recombinant mouse CD138 (R&D Systems) for 24 or 120 h. Phosphorylation of ERK was assessed in Western blot analysis, and the survival of cells was analyzed by FACS after DAPI staining. For B-cell differentiation analysis, B cells were first cultured in RPMI media containing 10% anti-CD138 beads pretreated MRL/Lpr mice serum with, and then were incubated with 250 ng/ml of APRIL and 10 ng/ml of LPS for 5 days. Stimulated cells were harvested, and percentages of plasma cells were assayed on DAPI-negative cells with FACS after staining with B220, CD138, and CXCR4 antibodies. Culture supernatants were also collected, and total or anti-dsDNA–specific IgM, IgG, and IgA antibody concentrations were determined in ELISA as described previously (26 (link)).