Welcome guest, Sign in

Home

X
加载中

Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. Recently, lipophilic cationic fluorescent dyes have been developed to detect Δψm by accumulating in the mitochondrial matrix until the Nernstian equilibrium distribution of lipophilic cations is reached. In this protocol, we applied a cell-permeant, green-fluorescent, lipophilic dye 3,3'-dihexyloxacarbocyanine Iodide (DiOC6(3)) which accumulates in mitochondria due to their large negative membrane potential, it can be applied to monitor the mitochondrial membrane potential using flow cytometric detection.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Flow Cytometric Detection of Mitochondrial Membrane Potential

Cell Biology > Cell-based analysis > Flow cytometry
Authors: Hsin-Yi Chang
Hsin-Yi ChangAffiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taipei
Bio-protocol author page: a571
Hsuan-Cheng Huang
Hsuan-Cheng HuangAffiliation: Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan
Bio-protocol author page: a572
Tsui-Chin Huang
Tsui-Chin HuangAffiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a573
Pan-Chyr Yang
Pan-Chyr YangAffiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan
Bio-protocol author page: a574
Yi-Ching Wang
Yi-Ching WangAffiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan
Bio-protocol author page: a575
 and Hsueh-Fen Juan
Hsueh-Fen JuanAffiliation: Department of Life Science, Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
For correspondence: yukijuan@ntu.edu.tw
Bio-protocol author page: a324
Vol 3, Iss 8, 4/20/2013, 8107 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.430

[Abstract] Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. Recently, lipophilic cationic fluorescent dyes have been developed to detect Δψm by accumulating in the mitochondrial matrix until the Nernstian equilibrium distribution of lipophilic cations is reached. In this protocol, we applied a cell-permeant, green-fluorescent, lipophilic dye 3,3'-dihexyloxacarbocyanine Iodide (DiOC6(3)) which accumulates in mitochondria due to their large negative membrane potential, it can be applied to monitor the mitochondrial membrane potential using flow cytometric detection.

Materials and Reagents

  1. Cells to analyze (this protocol has been successfully performed on A549, CL1-0, IMR-90, and MCF7 cells)
  2. Dulbecco's Phosphate-Buffered Saline (DPBS)
  3. DiOC6(3) (Life Technologies, Molecular Probes®, catalog number: D-273)
  4. DMSO (Sigma-Aldrich, catalog number: D8418)
  5. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148)
  6. 5 ml polystyrene BD falcon round-bottom tube with cell strainer cap (BD Biosciences, catalog number: 352235) 
  7. Sodium Chloride (NaCl)
  8. NaOH
  9. Potassium Chloride (KCl)    
  10. Potassium Phosphate, monobasic (KH2PO4)  
  11. Sodium Phosphate, dibasic (Na2HPO4)
  12. DPBS (see Recipes)
  13. DiOC6 stock (see Recipes)
  14. Paraformaldehyde stock (8%) (see Recipes)

Equipment

  1. Flow cytometry
  2. Water bath with temperature control
  3. Centrifuge
  4. 6-cm dish
  5. 0.22 μm filter

Procedure

  1. Cells were cultured with complete medium in a 6 cm dish at 37 °C and 5% CO2.
  2. Cells were harvested until 70-90% confluency reached.
  3. (For positive control of depletion of mitochondria membrane potential) Suspend 1 x 106 cells in 1 ml freshly prepared 4% paraformaldehyde diluted from stock using DPBS for 10 min at room temperature and wash them with 1 ml pre-warmed (37 °C) DPBS three times.
  4. Dilute the DiOC6 stock solutions into DPBS to make 0.1 μM working solution.
  5. Suspend cells at a density of 1 x 106 cells/ml in dye working solution and protect from light.
  6. Incubate the cells at 37 °C for 15 min.
  7. Centrifuge the tubes at 130 x g for 5 min.
  8. Remove the supernatant and gently resuspend the cells in 1 ml pre-warmed DPBS.
  9. Repeat the wash steps 7 and 8 twice.
  10. Submit samples to flow cytometry for mitochondrial membrane potential measurement.

Analysis

  1. Gate on the main cell population.



    Figure 1. Cells were analyzed according to their size and granularity. The X-axis represents the forward scatter (FSC) parameter which is relative to the size for the cell. The Y-axis shows the side scatter (SSC) parameter which correlates with the components inside the cell. Gate 1 indicates the main population of the cells we analyzed.

  1. Show the intensity of DiOC6(3) of cells in gate 1.


    Figure 2. Histogram of DiOC6(3). It shows how many cells are at each intensity of DiOC6(3). The X-axis represents the DiOC6(3) intensity, while the Y-axis indicates the cell counts in corresponding fluorescence intensity.

  1. Overlap the DiOC6(3) signals of positive control which indicating the population of depletion of mitochondria membrane potential (MMP).


    Figure 3. Overlapped histograms of healthy cells (red) and cells loss of mitochondria membrane potential (gray). DiOC6(3) histograms of healthy cells and cells loss of MMP were overlapped to compare the intensity differences.

  1. Gate on cells loss of MMP (R1) and healthy cells (R2).


    Figure 4. Selection of cell populations in health (R2) or depletion of MMP (R1). Healthy cells with high intensity in DiOC6(3) were gated in R1, while cells loss of MMP with lower DiOC6(3) intensity were gated in R1.

Recipes

  1. DPBS (1 L)
    8 g Sodium Chloride (NaCl)
    0.2 g Potassium Chloride (KCl)
    0.2 g Potassium Phosphate, monobasic (KH2PO4)
    1.15 g Sodium Phosphate, dibasic (Na2HPO4)
    Adjust to pH = 7.3.
  2. DiOC6 stock (10 mM)
    Dissolve 10 mg in 1.747 ml DMSO to make 1 mM stock.
    Aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  3. Paraformaldehyde stock (8%)
    Weight 8 g paraformaldehyde to 90 ml distilled water (in a fume hood).
    Add 0.1 ml of 10 N NaOH.
    Heat and stir the solution until the granules are fully dissolved (do not heat the solution above 65 °C).
    Turn off the heater and adjust to pH 7.4 with about 0.3 ml of 20% HCl.
    Bring volume to 100 ml with distilled water.
    Sterilize the solution with 0.22 μm filter and can be stored at 4 °C for 30-60 days.

References

  1. Chang, H. Y., Huang, H. C., Huang, T. C., Yang, P. C., Wang, Y. C. and Juan, H. F. (2012). Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein response. Cancer Res 72(18): 4696-4706.


How to cite: Chang, H., Huang, H., Huang, T., Yang, P., Wang, Y. and Juan, H. (2013). Flow Cytometric Detection of Mitochondrial Membrane Potential. Bio-protocol 3(8): e430. DOI: 10.21769/BioProtoc.430; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

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.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
12/2/2015 2:09:36 PM  

Rodrigo Hoyos
Boston Children's Hospital

Hi, I've been using this protocol in fibroblasts, but even thou the staining works I don't get a depletion of the mitochondrial membrane potential in the cells fixed with fresh 4% paraformaldehyde. If I compare the MFI of both the fixed and non fixed cells, the MFI is higher in the fixed ones, do you have any suggestions on how to solve this? Since the fixation did not work properly I analyzed the membrane potential comparing live and dead cells without fixation, but I'm not sure that's a proper control for the loss of membrane potential. I will be thankful for any help you could give me.

many thanks

Rodrigo

12/2/2015 5:23:34 PM  

Hsin-Yi Chang (Author)
Institute of Molecular and Cellular Biology,National Taiwan University

Hi Rodrigo,

Positive charged fluorescent dyes such as DiOC6 accumulates in mitochondria due to their large negative membrane potential. Therefore, the dye can not be retained in mitochondria after incubation of any reagent causes membrane permeabilization, e.g. aldehyde fixation or detergent permeabilization. Alternatively, you can treat cells with mitochondrial uncoupling agents for mitochondrial depolarization. We've routinely used the paraformaldehyde as the positive control for membrane potential depletion and works well. You can check whether you washed the cells thoroughly after paraformaldehyde treatment and DiOC6 staining, and used the same cell number for staining.

Hsin-Yi

Reply

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.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook
Other protocols by Hsin-Yi Chang(1)
Other protocols by Hsuan-Cheng Huang(1)
Other protocols by Tsui-Chin Huang(1)
Other protocols by Yi-Ching Wang(1)
Other protocols by Hsueh-Fen Juan(1)