DNA Staining for Cell Cycle Analysis

I. Sample

One or more of the following preparations:

  1. Suspension of single cells from tissue (e.g., lymph node, spleen, bone marrow, placental cells) 
  2. Tissue culture cells, including adherent cells detached from support 
  3. Ficoll-hypaque separated mononuclear cells 

II. Reagent - hypotonic staining buffer for DNA 

[Table to be inserted here]
Sodium citrate 
Triton–x 100 0.75ml 
0.25g 
Propidium iodide 0.025g 
Ribonuclease A 0.005g 
Distilled water 
250 ml 

We have kept this solution in a tightly-sealed bottle protected from light for several months without apparent loss of staining activity.

 III. Staining 

  1. Place 1x106 cells into each tube. 
  2. Spin down samples and remove supernatant as completely as possible without disturbing the pellet. 
  3. Add 0.5 mL of the hypotonic DNA staining buffer to the pellet and mix well. 
  4. Keep samples at 4°C protected from light for 15 min or for a maximum of 1 hr before acquisition on the flow cytometer. Note: Prolonged exposure to the hypotonic buffer can lead to an increase of debris in the sample and fragile cell types may only tolerate short exposure to the staining solution. 

Reference: Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66:188-193, 1975. 

Possible commercial sources:  

[Table to be inserted here]
Sodium citrate 
Triton-x 100 
Cat# C7254 Sigma-Aldrich, St. Louis, MO 
Cat# x100 Sigma-Aldrich, St. Louis, MO 
Ribonuclease A 
Propidium iodide 
Cat# R4875 Sigma-Aldrich, St. Louis, MO 
Cat# 537059 EMD Millipore, MA