5 m pore sized transwells

Chemotaxis assays were performed using 10⁶ BM or spleen cells incubated for 30 min with 1× DMEM containing 0.5% fatty acid–free BSA (EMD Biosciences), 5% of antibiotics, l-glutamine (Cellgro), and Hepes. Cells were then allowed to migrate through 5-µm-pore–sized transwells (Corning) toward soluble CXCL12 (R&D Systems), 2-AG (Cayman), or CXCL13 (PeproTech) for 3 h at 37°C. Cells were collected, stained, and resuspended in 40 ml of staining buffer and analyzed by flow cytometry for 40 s.

Supernatants for chemotaxis assays were prepared from confluent cell cultures. The media were replaced with DMEM containing 0.5% fatty acid–free bovine serum albumin (MP Biomedicals), 10 mM Hepes, antibiotics, and 2 mM l-glutamine for 10–12 h. Supernatants were collected and spun down to clean off contaminating cells. Chemotaxis assays were performed using primary murine BM cells or M12 B cell line transduced with an Ebi2 (IRES)-GFP retroviral construct (Kelly et al., 2011 (link)). Cells were allowed to migrate through 5-µm–pore sized transwells (Corning) for 3 h at 37°C with 5% CO₂ against supernatants collected from BMDM or OB cultures. Primary BM cells or M12 B cells were collected, stained with appropriate antibodies (BM cells), resuspended in 45 µl of staining buffer, and analyzed by flow cytometry for 40 s (LSRII; BD).