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Propidium Iodide Staining of Cells for FACS Analysis   

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Abstract

Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA staining can be used to study the cell cycle. Relative DNA content shows the proportion of cells in G1, G2 and S phases. Apoptotic cells show characteristic smear on DNA staining. Here a protocol to stain cells by PI is described.

Keywords: Propidium iodide, FACS, Cell cycle

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.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhu, H. (2012). Propidium Iodide Staining of Cells for FACS Analysis. Bio-protocol 2(11): e195. DOI: 10.21769/BioProtoc.195.
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I found some ref paper where they dis not use Triton X-100.
2/25/2013 5:46:34 PM Reply
Is it possible to do this experiment without Triton X-100?
2/25/2013 5:45:55 PM Reply
Hui Zhu
Stanford University

Triton X-100 is used as permeabilization buffer to penetrate cells. If you don’t penetrate cells, Propidium Iodide won’t stain well.

2/27/2013 1:55:48 PM


Roberta Ruela
Unicamp

You can also use a hypotonic buffer instead of Triton X-100 to permeabilize the cells.

9/4/2013 4:35:06 AM