MitoLite Orange 405 is a cationic dye that selectively accumulates in mitochondria probably vial the mitochondrial membrane potential gradient. The mitochondrial indicator is a hydrophobic compound that easily permeates intact live cells, and trapped in mitochondria after it gets into cells. This fluorescent mitochondrial indicator is retained in mitochondria for long time since the indicator carries a cell-retaining group. This key feature significantly increases its staining efficiency. The labeling protocol is robust, requiring minimal hands-on time. It can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, immunocytochemistry and flow cytometry. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. MitoLite Orange 405 is well excited by the violet laser at 405 nm, thus particularly useful for the flow cytometers with a violet laser source.