Triple Stained Endothelial Cells Revealing Cell Nuclei, Mitochondria, and the Actin Cytoskeleton

Three different fluorophores, MitoTracker Red CMXRos, BODIPY FL conjugated to phallacidin, and DRAQ5 were used to label an adherent culture of BPAE cells for mitochondria, the F-actin network, and nuclear DNA. The cells were first treated with the MitoTracker probe in growth medium for one hour, washed and fixed with paraformaldehyde (prepared in growth medium), permeabilized, and blocked with bovine serum albumen. The cells were then labeled with the conjugated phallacidin and counterstained with the anthraquinone (DRAQ5) reagent.