AAT Bioquest

How is propidium iodide typically applied to cells?

Posted July 24, 2023


Propidium iodide is typically applied to stain apoptotic cells for flow cytometry,
immunohistochemistry, and in situ hybridization techniques. The protocol for staining
cells with PI is described below.

Assay Protocol

  1. Cell Preparation: Dissolve the PI into a buffered solution such as PBS or Tris-EDTA
    buffer. The concentration of PI in staining solution may vary depending on
    experimental conditions, but is typically 1-5 mg/mL.
  2. Cell Harvesting: Cells are detached from the growth surface (e.g. culture dish) by
    using trypsinization for adherent cells, or centrifugation for suspension cells.
    Aliquot up to 1X10^6 cells/100 μL into polystyrene tubes and add 70% ethanol
    while gently vortexing.
  3. Cell Washing: Wash the cells by adding 2mL PBS, centrifuging at 300 X g for 5
    minutes and then pour out the buffer from pelleted cells. Repeat this step a total
    of 2 times to remove any residual media or serum components.
  4. Resuspension of cells: The staining solution containing PI (e.g. 0.1% BSA in PBS,
    Rnase, PI stock solution) should then be added to the cell suspension to achieve
    desired concentration of PI. Gently mix the cells and PI solution to ensure even
  5. Incubation of cells: Incubate the cells at 37 degrees celsius for a period of time
    depending on the experiment. Cells are typically incubated with PI for greater
    than 30 minutes in a dark room to be protected from light degradation.
  6. Analyzing cells: Following incubation, PI stained cells can be analyzed using flow
    cytometry, and are excited by a laser emitting light at a wavelength of 488 nm
    (blue-green). The emitted fluorescence is red in color and at a wavelength of 617
    nm. When used in IHC, PI is often used as a nuclear counterstain to visualize
    cellular nuclei within tissue sections.
Additional resources

Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays

Cell Viability Assays

Spectral Flow Cytometry

Propidium iodide *1 mg/mL aqueous solution*