Chemotaxis assays were performed using a transwell chamber (Costar) as previously described (1 (link)). Splenic B cells were immunostained for B cell subsets with fluorochrome-conjugated antibodies against B220, CD21, CD23, and CD24 washed twice, re-suspended in complete RPMI 1640 medium and added in a volume of 100 µl to the upper wells of a 24-well transwell plate with a 5 µm insert. Lower wells contained various doses of chemokines in 600 µl of complete RPMI 1640 medium. The numbers of cells that migrated to the lower well after 2 h incubation were counted using a MACSQuant flow cytometer (Miltenyi Biotec). The percent migration was calculated by the numbers of cells of a given subset that migrated into the bottom chamber divided by the total number of cells of that subset in the starting cell suspension, and multiplying the results by 100. D-erythro-sphingosine 1-phosphate was purchased from Avanti Polar Lipids. CXCL13, CCL19 and CXCL12 were purchased (R&D Systems). Fatty acid free bovine serum albumin (FAF-BSA) was purchased (Sigma-Aldrich).