Cell viability assay AO and PI

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Fluorometric cell viability assay using acridine orange and propidium iodide (AO/PI)

Read SDS in cupboard AO and PI are considered highly carcinogenic: use gloves and a face mask when preparing the concentrated stock solution, and use gloves when handling the working solution.


Using acridine orange (AO) and propidium iodide (PI) is a method to accurately assay viability of cells. AO and 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.


  • 5 μl Acridine Orange Stock (1 mg/ml or 1.4 mg/ml Acridine Orange hemi(zinc chloride) salt)
  • 3 μl Propidium Iodine Stock (1 mg/ml in H2O)
  • 1 ml PBS


AO/PI Working Solution

  1. Add 5 μl AO and 3 μl PI to 1 ml PBS and mix well
  2. Store at room temperature for up to 2 weeks

To Use

  1. Dilute cells with equal volume of AO-EB working solution
  2. Immediately look at cells under fluorescence microscope


  • Cells fluorescing orange, either fully or partially.= Dead
  • Green Cells = Alive