A fluorescense assay for micropinocytosis in isolated capillary endothelium

Micropinocytosis was quantitated in a suspension of capillary endothelium isolated from epididymal fat. A rhodamine-ferritin conjugate (RhF) was utilized as a tracer molecule for micropinocytic ingestion. RhF is electron dense, which permits ultrastructural determination of its mode of incorporation into endothelium, and its fluorescence allows quantitation of its uptake by micropinocytosis. The amount of RhF incorporated with time was standardized against endothelial cell DNA content as measured by fluorescence enhancement of mithramycin when bound to DNA. RhF was incorporated into isolated endothelium for periods exceeding 90 min.