B Cell Migration Assay Protocol

Fo B cells purified by magnetic-activated cell sorting were activated with 5 µg/ml goat anti–mouse IgM (Jackson ImmunoResearch) for 24 h or rested overnight (unstimulated control). Various dilutions of recombinant mouse CCL21 (provided by the late I. Clark-Lewis) or CXCL13 (PeproTech) in 150 µl chemotaxis buffer (RPMI-1640 with 0.5% BSA and 20 mM Hepes) were added to the lower chambers of Transwell chemotaxis plates (96-well, 5-µm pore size; Corning). Cells were extensively washed in chemotaxis buffer and loaded into the upper chambers at 10⁵ cells/well in 50 µl chemotaxis buffer and incubated for 3 h at 37°C. To enumerate B cell migration, cells were harvested from the bottom chambers, and B220⁺ cells were assessed by flow cytometry using a defined number of CaliBRITE beads (BD) as an internal reference. The migration index was calculated as described (Kara et al., 2013 (link)).