(A) Gating strategy for identifying different subsets of B cells from fetal, adult bone marrow, and umbilical cord blood. Cells were first gated based on forward and side scatter identifying the lymphocyte population. Staining for kappa and lambda light chains identified early (pro-B/pre-B), eg, κ⁻/λ⁻, and later (immature and mature B cells), eg, κ⁺/λ⁺, lineage B cells. Cells of the lymphocyte phenotype and negative for kappa/lambda immunoglobulin chains were further separated into CD19⁺, CD34⁺ pro-B cells and CD19⁺, CD34⁻ pre-B cells. Cells positive for kappa/lambda immunoglobulin chains were further identified as immature B cells (CD19⁺, IgD⁻) and mature B cells (CD19⁺, IgD⁺). (B) CXCR4 expression during B-cell development in bone marrow. Expression of CXCR4 on B-cells from adult (A), fetal (B) bone marrow, and umbilical cord blood (C) is shown. Cells were incubated with anti-CXCR4 specific antibody and analyzed by flow cytometry. Triple gates were set on different B-cell subsets and the percentage of CXCR4 positive cells was determined. The threshold line for positive cells was based on the maximum staining of a matched isotypic antibody with irrelevant specificity used in the same concentration as the anti-CXCR4 antibody. The isotype control antibody binding was analyzed on the same triple gated B-cell subsets as for CXCR4 expression. Anti-CXCR4 labeled cells that were brighter than those stained with the isotype control antibody were defined as positive for CXCR4. The shaded part of the histogram represents cells stained with anti-CXCR4 antibody, and the black line represents staining with the isotype control. The numbers represent the percentage of CXCR4 positive cells. The results are representative of 5 experiments using adult bone marrow B cells and 3 experiments each of fetal bone marrow and umbilical cord blood B cells.