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.
Red blood cells seen next to the arrows in the bright field image (above left) do not appear in the fluorescent image (above right).