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Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.

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[Bio101] Cell Cycle Analysis (PI) with GFP Detection

Cell Biology > Cell imaging > Fluorescence
Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
4/5/2012, 10744 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.199

[Abstract] Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.

Keywords: Cell cycle, GFP, FACS, Propidium iodide

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.


How to cite this protocol: Zhu, H. (2012). Cell Cycle Analysis (PI) with GFP Detection. Bio-protocol Bio101: e199. DOI: 10.21769/BioProtoc.199; Full Text



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6/8/2012 7:06:16 PM  

I have problems to detect the florescence of PI and GFP together, as their emission is very similar. Any suggestions?
Thanks.

6/10/2012 2:39:51 PM  

hui zhu
stanford universtiy

PI is much more broader which fluoresces everywhere from yellow to far-red.
You need to do compensation when go with GFP. You can use 7-AAD in place of
PI, which is more tighter.

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2/14/2012 12:21:55 PM  

Why glucose here?

2/22/2012 2:48:32 AM  

hui zhu
stanford universtiy

Paraformaldehyde is godd to penetrate cells. The disadvantage is that paraformaldehyde Only partially destroys osmotic properties of membranes. Osmolarity must be carefully adjusted by glucose or sucrose.

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Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
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