DNA Content/ Cell Cycle Analysis by Flowcytometry

  1. Prepare the fixative by filling a centrifuge tube with 4.5 ml of 70% ethanol. Keep the tube on ice.
  2. Collect cells and suspend 106 to 107 cells in 5 ml PBS in a centrifuge tube.
  3. Centrifuge cells (200 g, 6 min)
  4. Using a Pasteur pipette thoroughly resuspend cells in 0.5 ml PBS.*
  5. Transfer the cell suspension into the tubes containing 70% ethanol. Keep cells in fixative ≥2 hr.**
  6. Centrifuge the ethanol-suspended cells 5 min at 200 g. Decant ethanol thoroughly.
  7. Suspend the cell pellet in 5 ml PBS, wait 60 sec, and centrifuge (200 g, 5 min)
  8. Suspend cell pellet in 1 ml PI/Triton X-100 staining solution with RNase A. Keep either 15 min at 37°C or 30 min at room temperature.
  9. Perform flow cytometry 


*It is important to achieve a single-cell suspension. Fixation of cells that are in aggregates while suspended in PBS stabilizes the aggregates, which are then impossible to disperse. It is essential, therefore, to have a monodisperse cell suspension at the time of mixing cells with ethanol.

**Cells suspended in 70% ethanol can be stored at 0° to −40°C for several months if not years.



  • 70% ethanol
  • PBS
  • Propidium iodide (PI)/Triton X-100 staining solution with RNase A:

To 10 ml of 0.1% (v/v) Triton X-100 in PBS add 2 mg DNase-free RNase A and 200 μl of 1 mg/ml PI. Prepare freshly.

A stock solution of PI, made by dissolving 1 mg PI in 1 ml water, can be stored several months at 0° to 4°C.


Ref: Current Protocols in Cytometry, Unit 7.5