CB CD34⁺ cells that lack sLe^x epitopes have impaired binding to P- and E-selectin and are enriched in CD34⁺CD38^–/low cells. (A-C) Cells were incubated with PE-Cy5–conjugated anti-CD34, anti-sLe^x mAb HECA-452 identified with a PE-conjugated second antibody, and P-selectin/IgG identified with biotinylated protein A and streptavidin-FITC or E-selectin/IgM identified with a FITC-conjugated goat anti-IgM. Binding was measured by flow cytometry. The CB CD34⁺ cells were classified as sLe^x negative (R1 region, red) or sLe^x positive (R2 region, green) as shown in panel A. Cells in the gated R1 and R2 regions were examined for interactions with P-selectin and E-selectin as shown in panels B and C. (D-F) Cells were incubated with PE-Cy5–conjugated anti-CD34, PE-conjugated anti-CD38, and P-selectin or E-selectin identified as described in “Materials and methods”. The cells were gated as CD34⁺CD38^–/low (R1 region, red) and CD34⁺CD38⁺ (R2 region, green) as shown in panel D. Cells from the 2 gated regions were examined for interactions with P-selectin and E-selectin as shown in panels E and F. Cells incubated with isotype-matched control IgG or IgM were used to determine fluorescence thresholds for specific binding. The results are representative of 3 independent experiments.