How is propidium iodide typically applied to cells?

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
   staining.
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.