Immature B cells in the bone marrow do not efficiently enter blood and have elevated apoptosis in the absence of S1P1 receptor. (A–D) Mice were pulsed with BrdU, and B cells from the bone marrow (A and B) and peripheral blood (C and D) of control and B-S1pr1KO mice were analyzed by flow cytometry using anti-B220, anti-IgD, and anti-IgM antibodies in combination with BrdU detection methodology, as described in Materials and methods. Results are shown as dot plots (A and C), and as absolute numbers of BrdU⁺ B220^low IgM⁻ (pro–/pre–) and B220^low IgM⁺ (immature) B cells per femur (B) and BrdU⁺ B220⁺ IgD^low IgM^high (immature) and B220⁺ IgD^high IgM^low (mature) B cells per 400 µl of blood (D). The percentage of cells in each gate is indicated on the plots. Bars represent mean values, and the closed circles are individual mice. Data are representative of three experiments with three to five mice of each genotype per experiment. *, P < 0.05; **, P = 0.01 (Student's t test). (E and G) In vivo staining of bone marrow sinusoidal B cells. Mice were injected intravenously with PE-conjugated anti-CD45.2 antibody. After 2 min, the bone marrow cells and peripheral blood were obtained and stained with PerCP-conjugated anti-B220, APC-conjugated anti-IgM, and FITC-conjugated anti-IgD antibodies. Results are shown as histograms (E) and as the percentage of CD45.2-PE⁺ (sinusoidal cells; G) for pro–/pre– (B220⁺ IgD⁻ IgM⁻), immature (B220⁺ IgM⁺ IgD⁻ and B220⁺ IgM⁺ IgD^low), and mature (B220⁺ IgM⁺ IgD^high) bone marrow B cells from control and B-S1pr1KO mice. On the histograms (E), the bars show the percentage of PE-CD45.2⁺ cells for each group. (F) Lymphocytes stained in the peripheral blood by injection of PE-conjugated anti-CD45.2 antibody, showing equal staining in control and B-S1pr1KO mice. (H) Cells that were negative for anti-CD45.2–PE antibody staining were considered as parenchymal cells. Results are shown as mean values of four independent experiments (n = 10–12 mice per genotype). *, P < 0.05; **, P < 0.01; ***, P < 0.005 (Mann-Whitney test). (I and J) Annexin V staining was determined on B cells from the bone marrow of control and B-S1pr1KO mice. Results are shown as density plots (I) and as the percentage (J) of annexin V⁺ PI⁻ B220^low IgM⁻ (pro–/pre–) and B220^low IgM⁺ (immature) B cells per femur. Pooled data are from five experiments (n = 9 for each genotype). ***, P < 0.005 (Mann-Whitney test). (K and L) Annexin V staining on sinusoidal and parenchymal immature B cells. Cells were labeled in vivo with PE-conjugated anti-CD45.2 antibody and gated as in E and H. Annexin V staining was determined on immature bone marrow B cells (B220⁺ 7-AAD⁻ IgM⁺ IgD⁻ and B220⁺ 7-AAD⁻ IgM⁺ IgD^low) partitioned into parenchymal cells (CD45.2⁻; K) and sinusoidal cells (CD45.2⁺; L) from control and B-S1pr1KO mice. Pooled data are from three experiments (n = 8 for each genotype). *, P < 0.05 (Mann-Whitney test).