20µl of live cell sample and 20µl of AO/PI Staining Solution are combined. 20µl of stained sample is then added to a Cellometer Counting Chamber and analyzed in <60 seconds using a fluorescent Cellometer instrument

AO is permeable to both live and dead cells and stains all nucleated cells to generate green fluorescence. PI enters dead cells with compromised membranes and stains all dead nucleated cells to generate red fluorescence. Cells stained with both AO and PI fluoresce red due to quenching, so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.

Because mature mammalian red blood cells do not contain nuclei, only live and dead mononuclear cells produce a fluorescent signal. There is no need to lyse red blood cells, saving time and eliminating an extra sample preparation step.