Rcxcl12

We compared several protocols for BM-derived stromal cell (BMSC) generation and selected the one by Cen et al^{24 (link)} because it produced adequate stromal cell growth while minimizing immune cell contamination. Briefly, 20 × 10⁶ BM cells were plated (75-cm² flasks, complete OPTI media) with overnight adhesion with weekly media changes. CXCL12 and IL7 (R&D Systems) were analyzed by sandwich ELISA following the manufacturer’s protocol. Fluorescence-activated cell sorted (FACS) pro-B cells (CD19⁺IgD⁻IgM⁻B220⁺CD43⁺) from BM were cultured in a 96-well plate (5 × 10³ to 10 × 10³ cells per well) in complete OPTI media containing 1/200 IL7 (a gift from Christopher Paige, University Health Network, Toronto, ON, Canada) and conditioned media from plates of equally numbered BMSCs. Cells were collected after 6 days for analysis. For B-cell migration, 10⁶ BM cells were plated on the upper Transwell chamber (6.5-mm insert; 5-μm pore polycarbonate membrane) in a 24-well plate, with media plus 100 ng/mL recombinant CXCL12 (rCXCL12; R&D Systems) or BMSC culture supernatant in the bottom chamber. Cells were analyzed by a flow cytometer after 3 hours.