Integrin-Mediated Cell Adhesion Assay

96-well plates (Nunc) were coated with human sCD40L (Biozol, Germany) or human fibrinogen, ICAM-1, heparin, RAGE, vitronectin, JAM-C, or NIF (10 μg/ml, all from R&D Systems, USA) and incubated with CHO cells expressing constitutively activated, human Mac-1 (Mac-1 del) or CHO cells expressing the naïve, non-activated Mac-1 (Mac-1 WT)^{43 (link)}. As controls, CHO cells expressing no integrin (CHO) were used. Cells were pre-incubated with blocking antibodies (10 μg/ml) for 15 min and allowed to adhere for 50 min. Adhering cells were counted after repeated washing with PBS. Alternatively, we tested the adhesion of primary mouse peritoneal macrophages to mouse CD40L (R&D Systems, USA). For dynamic adhesion assays, mouse endothelial cell cultures (HUVECs) were grown to confluency in 35 mm cell culture dishes as previously described^{41 (link)}, stimulated with TNFα overnight, and placed in a parallel flow chamber system (Glycotech). The number of adhering cells was quantified at the indicated shear rate in the presence of the indicated antibodies (10 μg/ml).