For viability determination, 20 µl of sample is mixed with 20 µl of Cellometer AO/PI Staining Solution. The acridine orange (AO) dye stains DNA in all nucleated cells, generating green fluorescence. Propidium iodide (PI) stains DNA in all cells with compromised cell membranes, generating red fluorescence. In cells stained with both AO and PI, the green fluorescence is absorbed by the red fluorescence via FRET (fluorescence resonance energy transfer). All dead nucleated cells fluoresce red (image far right). All live nucleated cells fluoresce green (image right). Live and dead cells are counted in the fluorescent images.
Red blood cells (RBCs), platelets, and debris do not fluoresce and are excluded from cell counts, so sample lysis is not required with the AO/PI viability method.