Microtiter assays for quantitation of assembly of plasma and cellular fibronectin

Fibronectin (FN) is a plasma glycoprotein produced by hepatocytes that circulates at near micromolar concentration and assembles into extracellular matrix fibrils at cell surfaces along with locally produced cellular FN. We describe two microplate assays that quantify assembly of human FN by cells in monolayer culture. One assay measures fluorescence due to incorporation of ALEXA488-plasma FN into matrices of fibroblasts and has been used successfully in high-throughput screens. The other measures fluorescence due to binding of fluorochrome-labeled antibody to the EDA domain of cellular FN synthesized and deposited by various cell types. The assays take advantage of the tight association of assembled FN with cell monolayers and adherence of cell monolayers to wells of microtiter plates. The assays are straightforward, adapt to 96- and 384-well formats, and use reagents that are also suitable to image FN that is assembled into fibrils.