Studies of fibronectin matrices in living cells with fluoresceinated gelatin

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC-gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC-gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC-gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.