Esterase-catalyzed hydrolysis of Sun Red acetate (SRA) yields the Sun Red fluorophore that can be excited with the 633 nm laser with emission of ~660 nm. The fluorescence of Sun Red can be readily detected using the Cy5 filter set that is commonly equipped with most of the commercial fluorescence instruments. Although Sun Red is readily excited at 633 nm with red emission of ~660 nm, SRA has very minimal absorption at 633 nm without red emission, making SRA one of the most sensitive NIR esterase substrates. SRA provides a second color for cell viability assay while the popular fluorescein color can used for another cellular functional assay. SRA is a non-fluorescent hydrophobic compound that can pass through the cell membrane whereupon intracellular esterases hydrolyze the acetate group producing the highly fluorescent product Sun Red. The Sun Red molecule accumulates in cells that possess intact membranes so the deep red fluorescence can be used as a marker of cell viability. Cells that do not possess an intact cell membrane or an active metabolism may not accumulate the fluorescent product and therefore do not exhibit deep red fluorescence. SRA may be used in combination with a green vital stain such as a FITC or Alexa Fluor 488-labeled antibody.
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