Cell surface binding of recombinant OPG. (A) surface expression of TRAIL and RANKL in PMNs, HL60 cells, and HUVECs was evaluated by flow cytometry. The control (unshadowed) histograms represent the background fluorescence obtained from the staining of the same cultures with isotype-matched control antibodies. One of 4 experiments with similar results is shown. (B) HL60 and HUVECs were incubated with either recombinant OPG or OPG-Fc, and binding to cell surface was revealed by flow cytometry. Results are expressed as mean fluorescence intensity and are mean ± SD of determinations of 3 separate experiments. (C) The specific contribution of heparan sulfate proteoglycans in OPG-mediated leukocyte adhesion was evaluated by pretreatment of OPG with heparin or pretreatment of HUVECs with heparinase I, II, and III (Hep.) plus Chondroitinase (Chond.) before adhesion assays. Values are mean ± SD of triplicate determinations of 3 separate experiments. ∗, P > .05.