ViaStain™ AO/PI Viability Stains

AO/PI Staining Solution: For accurate determination of live / dead nucleated cell concentration in heterogeneous samples using dual-fluorescence.

Dual-Fluorescence Viability, using acridine orange (AO) and propidium iodide (PI), is the recommended method for accurate viability analysis of primary cells, such as PBMCs, splenocytes, and stem cells in samples containing debris and unwanted non-nucleated cell types including red blood cells.

Acridine orange (AO) and propidium iodide (PI) are nuclear staining (nucleic acid binding) dyes. 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.

For AO/PI staining and viability determination, 20 µl of live cell sample and 20 µl 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. Each instrument automatically reports live / dead cell number, live/dead cell concentration, mean diameter, and percent viability for the sample tested.

ViaStain™ Calcein AM

Calcein AM (Calcein acetoxymethyl ester) is a cell permeable, non-fluorescent compound. Upon crossing the cell membrane, calcein-AM is rapidly hydrolyzed by cellular esterases inside live cells. The hydrolysis cleaves the AM group, converting the non-fluorescent calcein AM to a strongly green fluorescing calcein. Because the created calcein is more hydrophilic it is trapped inside the cells with intact membranes. Cells that do not possess active cytoplasmic esterases are unable to convert calcein AM to calcein, and therefore do not fluoresce green. This allows for a quick and easy detection of metabolically-active cells in a sample.

Since calcein-AM does not require DNA binding, it stains all metabolically-active cells and can be used to measure metabolic activity in non-nucleated cells, such as platelets. Because calcein AM is photostable, has low cytotoxicity and does not affect cellular functions, it is a popular stain for the examination of cell vitality and metabolic activity.

ViaStain™ Calcein-AM / PI Cell Viability Kit

The Nexcelom Calcein-AM/PI Cell Vitality and Viability kit enables measurement of the number and concentration of both metabolically active and dead, or non-viable, cells in a population. The software automatically calculates percent vitality/viability based on the number of Calcein-positive (metabolically active) cells, and the number of PI-positive (non-viable) cells.

Assay Principle

Acetoxymethyl ester of calcein (calcein-AM) is a non-fluorescent dye that passively enters cells. In metabolically active cells, the dye is converted by cytosolic esterases into green fluorescent calcein. The fluorescent calcein is retained by cells with intact membranes. Metabolically-active cells fluoresce in the green channel. Propidium iodide (PI) is a DNA-binding dye that enters cells with compromised membranes. Dead (compromised) nucleated cells fluoresce in the red channel.