The following protocol is for DNA staining with propidium iodide for cell cycle analysis (complex hypotonic solution). 

Reagents

Stock Solution

  • 20mg/ml propidium iodide (in 1X PBS) [store refrigerated, protected from light]

10X Lysis Buffer

  • 10mM TRIS (1.211g/L in dd H2O) [MW=121.1g]
  • 1mM EDTA (0.3722g/L in dd H2O) [EDTA Disodium Sulfate2H2O MW= 372.2g]
  • 1% Triton X-100
  • Store at room temperature

Hypotonic Staining Solution (for 50ml)

  • 50mg sodium citrate
  • 5ml lysis buffer
  • 45ml ddH2O
  • Mix until sodium citrate dissolved
  • 125µl stock propidium iodide solution
  • Store refrigerated and protected from light [good for at least 2 weeks]

Staining

  1. Pellet 2 x 106 cells.
  2. Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
  3. Add 1ml hypotonic propidium iodide staining solution.
  4. Vortex.
  5. Store on ice for at least 15 minutes before running on flow cytometer.

Important

The number of cells in staining solution should be kept at a constant ratio of 1 x 106 cells per ml of hypotonic PI staining solution. If you have more or less cells than the recommended number, adjust staining volume appropriately.

For Adherent Cell Cultures

Cells should be harvested using a method that yields viable, single cells in suspension. The most common harvest method is by trypsinization. Use what ever method that you feel yields the best results. Once a good quality cell suspension has been established then stain the cells using the method outlined above. Adherent cell lines often do not lyse as completely as non adherent cell lines, leaving part of the RNA rich cytoplasm attached to the nucleus. Since PI stains both RNA and DNA, it may be necessary to add RNAase to the PI solution to eliminate the residual RNA. It is very important that a high quality suspension of stained, single nuclei be established before attempting analysis on the flow cytometer. Some cell lines can be very difficult to prepare successfully. Therefore, it is highly recommended that nuclei suspensions be examined under a fluorescent microscope to determine their quality before running them on the flow cytometer. The Flow Cytometry Facility has a fluorescent microscope available for this purpose.

An alternative harvest method to trypsinization is to add hypotonic PI solution directly to the cell flask. A reasonable estimation of the cell numbers in the flask should be determined first so that the appropriate volume of hypotonic PI solution can be added.