WASP-deficient MZ B cells show impaired in vitro migration to S1P. (A) Flow cytometric analysis of cell death in freshly isolated FO and MZ B cells, identified based on CD21 and CD23 expression. Necrotic cells were defined as 7-AAD⁺ (left panel) and apoptotic cells as 7-AAD⁻ annexin V⁺ (right). N = 6 mice per group. (B) Adhesive response of T1, FO, T2-MZP, and MZ B cells to ICAM-1 (left panel) and VCAM-1 (right). Splenocytes were allowed to adhere to tissue culture plates coated with indicated ligands for 2 hours. Adherent cells were released by ethylenediaminetetraacetic acid, stained for CD21, CD23, and IgM to define B-cell subsets, and enumerated by flow cytometry. The percentage of adhesive cells as a fraction of total input cells is shown as mean values (± SD). This experiment is representative of 2 similar ones with n = 3 mice per group. (C) Migratory response of MZ B cells to S1P was determined by an in vitro chemotaxis assay. The percentage of cells that migrated after 3 hours in the chemotaxis assay was determined and represents the mean value (± SD) of n = 3 mice per group. This experiment represents 2 similar experiments. (D) Real-time PCR analysis of S1P₁, and S1P₃ in FO, T2-MZP, and MZ B cells. B cells from 3 mice per group were pooled and cell-sorted based on CD21, CD23, and IgM expression. Samples were run in triplicate, and the target mRNA was normalized to HPRT mRNA. The mRNA content of test sample in WT FO B cells was defined as 1 arbitrary unit. Mean value (± SD) of triplicates is shown, and the data are representative of 3 similar experiments. *P < .05, **P < .01.