The University of Iowa Carver College of Medicine

Flow Cytometry Facility

DNA Staining with PI

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

This recipe is for a 50µg/ml propidium iodide (Sigma) in 0.1% sodium citrate solution.

For 500ml

  • 0.025gm PI + 0.5gm sodium citrate + 500ml 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 the 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 propidium iodide 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.