Using Acridine Orange/Propidium Iodide (AO/PI) to Measure Cell Viability
Acridine orange (AO) and propidium iodide (PI) are nucleic acid binding dyes that can be used to measure the cell viability. Both AO and PI are considered skin irritants and may be harmful if ingested or inhaled; they are currently not classified as carcinogenic. Since AO is cell permeable, all stained nucleated cells generate a green fluorescence. PI (~668 Daltons) only enters cells with compromised membranes and therefore dying, dead, and necrotic nucleated cells stained with PI generate a red fluorescence. When cells are stained with both AO and PI, live nucleated cells only fluoresce green and dead nucleated cells only fluoresce red. This is due to Förster resonance energy transfer (FRET); where the PI signal absorbs the AO signal producing no spill-over or double positive results. Additionally, other membrane-exclusion viability dyes such as: ethidium bromide (EB), 7AAD, SYTOX green/red, DRAQ5 and others may also be used instead of PI. Cell viability is calculated by examining the ratio of the number of live to the number of dead fluorescing cells. This assay can not only be used to measure the viability of nucleated cells in cell culture and purified samples but also in complicated samples such as PBMC, whole blood, bone marrow, bronchoalveolar lavage, tumor digests, primary samples and many more.