What is the concept behind the Cyto3D® Live-Dead Assay Kit co-stain?

When both AO and PI are used to stain cells, live nucleated cells exclusively emit a green fluorescence, while deceased nucleated cells produce a red fluorescence. This is due to Förster resonance energy transfer (FRET), where the PI signal absorbs the AO signal, resulting in no cross-contamination or double-positive outcomes.

Acridine orange (AO) and propidium iodide (PI) are dyes that bind to nucleic acids and are employed for assessing cell viability. It’s important to note that AO and PI are categorized as skin irritants and should be handled carefully to avoid ingestion or inhalation. They are, however, not currently classified as carcinogenic substances.

AO can penetrate cell membranes, causing all nucleated cells that it stains to emit a green fluorescence. On the other hand, PI, with a molecular weight of approximately 668 Daltons, can only enter cells with compromised membranes. Consequently, stained with PI, nucleated cells undergoing death or necrosis exhibit a red fluorescence.

When both AO and PI are used to stain cells, live nucleated cells exclusively emit a green fluorescence, while deceased nucleated cells produce a red fluorescence. This is due to Förster resonance energy transfer (FRET), where the PI signal absorbs the AO signal, resulting in no cross-contamination or double-positive outcomes.

Cell viability is determined by assessing the ratio of live to dead fluorescing cells. This assay is versatile, finding applications not only in gauging the viability of nucleated cells in cell culture and purified samples but also in complex specimens like peripheral blood mononuclear cells (PBMC), whole blood, bone marrow, bronchoalveolar lavage, tumor digests, primary samples, and more.