Establishment of a standardized assay system of fibronectin activity using fibronectin-mediated cell adhesion

An in vitro assay of fibronectin (FN) was established based on the adhesion of baby hamster kidney (BHK) cells through the cell-binding domain of FN. Each well of a microtiter plate was coated with samples or various concentrations of standard FN. Bovine serum albumin mas further coated to prevent the non-specific adhesion of the cells. Various numbers of BHK cells mere plated and incubated, After mashing out the non-attached cells, the number of attached cells was measured using neutral red (NR)-staining. The conditions for the assay were optimal when 1 x 10(5) cells/well mere plated and incubated for 90 min. The linear relationship between the concentration of FN coated and the absorbance of IVR was observed in the range of 0.1-1.0 mu g/ml of FN. The inhibition of cell binding by the peptides containing an Arg-Gly-Asp (RGD) sequence demonstrated that this assay system depended on FN-mediated cell adhesion through the major cell-binding domain.