Propidium Iodide Staining of Cells for FACS Analysis    

Materials and Reagents

1. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
   
2. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
   
3. RNase A (Sigma-Aldrich, catalog number: R4642 )
   
4. Phosphate buffered saline (PBS)
   
5. 70% EtOH
   
6. 10 % Triton X-100 (see Recipes)
   
7. PI stock solution (see Recipes)
   

Equipment

1. Standard table-top centrifuges
   
2. FACS machine
   
3. Incubator
   

Procedure

1. Trypsinize and harvest cells, fix cells into 0.5 ml 70% EtOH (pre-cooled to -20 °C overnight).
   
2. Store fixed cells on ice at least 1 h and for up to several days.
   
3. Spin down cells for 2 min at 4,000 rpm.
   
4. Resuspend cell pellet in 0.5 ml PBS containing 0.25% Triton X-100 and incubate on ice for 15 min.
   
5. Spin down the cells for 2 min at 4,000 rpm.
   
6. Discard supernatant and resuspend cell pellet in 0.5 ml PBS containing 10 μg/ml RNase A and 20 μg/ml PI stock solution, transfer to FACS tubes and incubate at room temperature (RT) in the dark for 30 min.
   
7. Ready for FACS.
   

Recipes

1. 10% Triton X-100
   1 ml Triton X-100
   9 ml ddH₂O
   
2. 1 mg/ml PI stock solution
   10 mg PI
   10 ml ddH₂O
   

Acknowledgments

This protocol was developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

1. Seki, A., Coppinger, J. A., Jang, C. Y., Yates, J. R. and Fang, G. (2008). Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320(5883): 1655-1658.
   
2. 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.