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 adh esion of baby hamster kidney (BHK) cells through the cell-binding doma in of FN. Each well of a microtiter plate was coated with samples or v arious concentrations of standard FN. Bovine serum albumin mas further coated to prevent the non-specific adhesion of the cells. Various num bers of BHK cells mere plated and incubated, After mashing out the non -attached cells, the number of attached cells was measured using neutr al 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 r elationship 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 inhibit ion of cell binding by the peptides containing an Arg-Gly-Asp (RGD) se quence demonstrated that this assay system depended on FN-mediated cel l adhesion through the major cell-binding domain.