Cremaster muscle endothelial cells bind L-selectin. (A) L-selectin–IgG binding to neutrophils. Peripheral blood leukocytes from WT, PSGL-1^−/−, and FucT-IV^−/−/VII^−/− mice treated with or without an intrascrotal injection of TNF-α were stained with L-selectin–IgG complexed with PE-labeled goat F(ab′)₂ anti–human IgG with (shaded histograms) or without (open histograms) EDTA, and then stained with anti–Gr-1-APC. Cells were gated for neutrophils (SSC^highGr-1^high). (B) Expression of endothelial markers on cremaster muscle endothelial cells. Cremaster muscle was removed from WT mice treated with or without an intrascrotal injection of TNF-α. Single-cell suspensions enriched for endothelial cells by Percoll were stained with the indicated mAbs (open histograms) or isotype controls (shaded histograms) followed by secondary reagents together with anti–CD31-APC and anti–CD45-FITC or -PE-Cy5. Cells were gated on CD31⁺CD45⁻, as shown in the top panel. (C-E) L-selectin–IgG binding to cremaster muscle endothelial cells. Single-cell suspensions from the cremaster muscle were stained with L-selectin–IgG complexed with PE-labeled goat F(ab′)₂ anti–human IgG and then stained with anti–CD31-APC and anti–CD45-FITC. Cells were gated on CD31⁺CD45⁻. (C) L-selectin–IgG binding to cremaster muscle endothelial cells from WT, PSGL-1^−/−, and FucT-IV^−/−/VII^−/− mice treated with or without an intrascrotal injection of TNF-α. The cells were stained with (shaded histograms) or without (open histograms) EDTA. Percentages of L-selectin–binding cells are given. The numbers in parentheses indicate mean fluorescence intensity values. (D) Inhibition of L-selectin–IgG binding to cremaster muscle endothelial cells by the anti–L-selectin mAb MEL-14. The L-selectin–IgG secondary antibody complex was incubated with MEL-14 (dotted line) or isotype control (solid line) before added to the cells. (E) L-selectin–IgG binding to cremaster muscle endothelial cells treated with heparitinase. The cells were treated with heparitinase before being analyzed for L-selectin–IgG binding. The cells were stained with (shaded histograms) or without (open histograms) EDTA. The cells were also stained with (open histograms) or without (shaded histograms) the anti–Δ-heparan sulfate mAb 3G10 followed by FITC-labeled donkey anti–mouse IgG. Results represent one of 3 to 5 similar experiments.