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Cell Cycle Analysis Using Propidium Iodide Staining with GFP Detection   

Abstract

Materials and Reagents

1. Phosphate buffered saline (PBS)
   
2. Glucose (Sigma-Aldrich, catalog number: G8270 )
   
3. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15170 )
   
4. 70% EtOH
   
5. Hepes (Sigma-Aldrich, catalog number: H3375 )
   
6. NP-40 (MBL International, catalog number: JM-2111-100 )
   
7. BSA (Sigma-Aldrich, catalog number: A3803 )
   
8. RNase A (Sigma-Aldrich, catalog number: R4642 )
   
9. Fix solution (see Recipes)
   
10. Wash solution (see Recipes)
    

Equipment

1. Centrifuges (Beckman Falcon, TLS-55 )
   
2. 15 ml polypropylene falcon tubes (BD Biosciences, Falcon^®, catalog number: 352097 )
   
3. FACS machine
   

Procedure

1. Trypsinize and harvest cells, suspend in 10 ml PBS into 15 ml polypropylene falcon tubes.
   
2. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
   
3. Wash cells 1x in 5 ml PBS.
   
4. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
   
5. Thoroughly resuspend cells 1 ml fix solution.
   
6. Incubate 10 min on ice.
   
7. Add PBS up to 14-15 ml.
   
8. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
   
9. Wash cells 1x in 5 ml PBS.
   
10. Resuspend cells in 0.5 ml 1x PBS. Vortex tube gently and add 4.5 ml ice cold 70% EtOH dropwise over 30 sec to 1 min. Incubate on ice > 1 h.
    
11. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
    
12. Wash cells 1x in wash solution.
    
13. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
    
14. Resuspend cells in 500 μl of PBS containing 10 μg ml^-1 RNase A and 20 μg ml^-1 PI, transfer to FACS tubes and incubate at room temperature in the dark for 30 min.
    
15. FACS immediately or store at 4 °C until FACS analysis.
    

Recipes

1. Fix solution
   1x PBS
   2% Glucose
   3% Paraformaldehyde
   
2. Wash solution
   1x PBS
   20 mM Hepes
   0.25% NP-40
   0.1% BSA
   

Acknowledgments

This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.

References

1. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.