Competition of SSL5 with anti–PSGL-1 mAbs binding to neutrophils. (A) Neutrophils were incubated with a concentration range of SSL5 (0.3-10 μg/mL) for 30 minutes on ice. After washing, the cells were treated with 1 μg/mL anti–PSGL-1 PL1, PL2, or KPL1, or 1 μg/mL isotype control anti-C5aR W17/1. Bound antibodies were detected with FITC-conjugated goat anti–mouse IgG. Data represent relative binding of PL1 (▪), PL2 (▴), KPL1 (▾), or W17/1 (♦) to cells treated with SSL5 compared with control-treated cells and are mean values ± SEMs of 3 independent experiments. (B) Representative histograms of panel A depict binding of 1 μg/mL PL1, PL2, KPL1, and W17/1 to neutrophils pretreated without (thin continuous line) or with (thick continuous line) 10 μg/mL SSL5. Gray histograms represent cells stained only with the secondary antibody. (C) Experiment as described in Panel A using SSL7 instead of SSL5. Data represent relative binding of PL1 (▪), PL2 (▴), KPL1 (▾), or W17/1 (♦) to cells treated with SSL7 compared with control-treated cells and are mean values ± SEMs of 3 independent experiments. (D) Two-color flow cytometry was used to analyze binding of 1 μg/mL KPL1 to different leukocyte subpopulations in the absence (▪) or presence (□) of 10 μg/mL SSL5. Neutrophils were selected on gating, whereas monocytes, T lymphocytes, and B lymphocytes were stained with anti–CD14-PE, anti–CD4-PE or anti–CD8-PE, and anti–CD19-PE, respectively. NK cells were first negatively selected for anti–CD3-Cy and then positively selected for anti–CD16-PE and anti–CD56-PE. The data represent mean fluorescence of detected KPL1 and are mean values ± SEMs of 3 independent experiments.