Static adhesion of leukocytes to immobilized VWF. (A) Freshly isolated PMNs (2 × 106 cells/mL) were incubated with PMA (100 nM) for 15 minutes and added to immobilized VWF, fibrinogen, or BSA at a concentration of 1 × 105 cells/well for 30 minutes at room temperature. After washing, adherent cells were visualized by light microscopy (original magnification, 400×). Freshly isolated PMNs (B), monocytes (C), THP-1 cells (D), and U937 cells (E) were stimulated with PMA for 15 minutes and added at a concentration of 1 × 105 cells/well to immobilized VWF, fibrinogen, or BSA for 30 minutes at room temperature (B-C) or for 60 minutes at 37°C (D-E). To quantify adhesion, bound cells were lysed using 1% Triton-X100 and 50 mM acetic acid (pH 5.0), and endogenous alkaline phosphatase activity was determined using PNP as a substrate. Relative adhesion is plotted with adhesion to VWF as a reference (100%) in each panel. The number of adhered cells was dependent on the cell type used and varied between 10% (U937 cells) and 40% (PMNs) of the total cells added. Data are corrected for adhesion to uncoated wells (less than 5% of VWF-coated wells) and represent the mean ± SEM of 3 to 8 experiments performed in duplicate.
