The following protocol is for DNA Staining with propidium iodide for cell cycle analysis (detergent hypotonic solution)

Used the same as the plain hypotonic propidium iodide solution, it renders better staining for some cells. The following recipe is for a 50µg/ml propidium iodide (Sigma) and 0.1% Triton X-100 in 0.1% sodium citrate solution.

For 500ml

• 0.025gm PI + 0.5gm sodium citrate + 0.5ml Triton X-100 +499.5ml ddH₂O
• Store refrigerated and protected from light.

Staining Cells

1. Pellet 5 x 10⁵ cells.
2. Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
3. Add 0.5ml cold, hypotonic propidium iodide solution.
4. Vortex vigorously.
5. Store on ice for at least 15 minutes before running on the flow cytometer.
6. The nuclei suspension should remain on ice until run.

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.