AO/PI Viability

AO/PI Viability: Dual-fluorescence for live/dead nucleated cell concentration in heterogeneous samples

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, such as 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 Förster resonance energy transfer (FRET), so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.

Live nucleated cells stained with Acridine Orange

Live nucleated cells stained with Acridine Orange

Dead nucleated cells stained with Propidium Iodide

Dead nucleated cells stained with Propidium Iodide

Because mature mammalian red blood cells don’t 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. Red blood cells seen next to the arrows in the bright field image (below left) do not appear in the fluorescent image (below right).

only live and dead mononuclear cells produce a fluorescent signal

Nexcelom has evaluated the AO/PI method with a wide range of primary cells from peripheral blood, cord blood, bone marrow, tissue, and other sample types. The Cellometer Auto 2000 Cell Viability Counter, Cellometer Vision Cell Analyzer, and Cellometer Vision CBA Analysis System include pre-optimized assays for analysis of AO/PI Viability.

By |2012-06-14T11:10:15+00:00June 14th, 2012|Categories: About Cell-Based Assays|2 Comments


  1. Ritam Chatterjee June 4, 2015 at 11:09 pm - Reply

    I have tried DAPI/PI staining to detect apoptosis in bone marrow cells but with the addition of PI into the sample whole matter became a mass and ultimately I could`nt see anything under fluorescence microscope. Can you suggest me the exact protocol? I shall be highly obliged by it.

  2. Leo Chan
    Leo Chan June 24, 2015 at 8:36 pm - Reply

    Hi Ritam, DAPI and PI are both viability stains, they will not detect specifically apoptosis in the cells. One other thing is, are your bone marrow cells purified?

    To measure apoptosis, you have to use Annexin V/PI or Caspase fluorescent methods.

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