Welcome guest, Sign in

Home

X
加载中

Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.

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 Reactive Oxygen Species

Cell Biology > Cell-based analysis > Flow cytometry
Authors: Hsin-Yi Chang
Hsin-Yi ChangAffiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
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 Pharmacology, National Cheng Kung University, Tainan, 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, 12712 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.431

[Abstract] Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.

Materials and Reagents

  1. Cells to analyze (this protocol has been successfully performed on A549, CL1-0, and IMR-90 cells)
  2. Dulbecco's Phosphate-buffered saline (DPBS)
  3. 2’,7’-dichlorofluorescein diacetate (H2DCFDA) (Life Technologies, InvitrogenTM, catalog number: D-399)
  4. Anhydrous DMF (N,N-dimethylformamide) (Sigma-Aldrich, catalog number: 227056)
  5. N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, catalog number: A7250)
  6. Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 349887)
  7. 5 ml polystyrene BD falcon round-bottom tube with cell strainer cap (BD Biosciences, catalog number: 352235)
  8. Sodium Chloride (NaCl)                     
  9. Potassium Chloride (KCl)                                       
  10. Potassium Phosphate, monobasic (KH2PO4)                                   
  11. Sodium Phosphate, dibasic (Na2HPO4)
  12. DPBS (see Recipes)
  13. H2DCFDA stock (10 mM) (see Recipes)
  14. NAC stock (1 M) (see Recipes)
  15. H2O2 stock (1 M) (see Recipes)

Equipment

  1. Flow cytometry
  2. Water bath
  3. Centrifuge

Procedure

  1. Cells were cultured with complete medium in a 6 cm dish at 37 °C and 5% CO2.
  2. Cells were treated with NAC or H2O2 after reaching 70-90% confluency. ROS scavenger NAC is a precursor to cysteine and glutathione which are strong antioxidants; while H2O2 is a compound with an oxygen-oxygen single bond and is known as a strong oxidizer.
  3. (For negative control) Incubate cells with freshly prepared 5 mM NAC in culture medium for 1 h at 37 °C after three time washes of pre-warmed DPBS.
  4. (For positive control) Incubate cells with freshly prepared 0.1 mM H2O2 from 1 M stock in DPBS for 20 min at 37 °C after three time washes of pre-warmed DPBS.
  5. Dilute the H2DCFDA stock solutions into DPBS to make 0.1 μM working solution.
  6. Cells were harvested by trypsinization.
  7. Suspend cells in working solution at a density of 1 x 106 cells/ml and incubate at 37 °C for 30 min and protect from light.
  8. Centrifuge the tubes at 130 x g for 5 min.
  9. Remove the supernatant and gently resuspend the cells in pre-warmed DPBS.
  10. Repeat the wash steps 8 and 9 twice.
  11. Submit samples to flow cytometry for ROS detection using the 488 nm laser for excitation and detected at 535 nm.

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 H2DCFDA of cells in gate 1.



    Figure 2. Histogram of H2DCFDA. It shows how many cells are at each intensity of H2DCFDA. The X-axis represents the H2DCFDA intensity, while the Y-axis indicates the cell counts in corresponding fluorescence intensity.

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. H2DCFDA stock (10 mM)
    Dissolve 10 mg in 2.05 ml DMF to make 10 mM stock.
    Aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  3. NAC stock (1 M)
    Dissolve 1 g in 6.128 ml distilled water to make 1 M stock.
    Filter, aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  4. H2O2 stock (1 M)
    Dilute 50 μl H2O2 from 35% (=11.6M) in 530 μl sterile deionized water to make 1 M stock.

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 this protocol: Chang, H., Huang, H., Huang, T., Yang, P., Wang, Y. and Juan, H. (2013). Flow Cytometric Detection of Reactive Oxygen Species. Bio-protocol 3(8): e431. DOI: 10.21769/BioProtoc.431; 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
12/3/2015 8:29:54 AM  

Rodrigo Hoyos
Boston Children's Hospital

Hi, I'm trying to set up your protocol and I wanted to confirm if NAC/H2O2 were present in the cell culture media while the cells were getting to a confluency of 70-90% (for positive and negative controls respectively) and after cells were treated for an adittional hour. (Steps 2, 3 and 4 of your protocol). Thanks for your help.

12/4/2015 3:32:51 AM  

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

Hi, I'm not sure what your question is. According to the protocol, cells were treated with NAM or H2O2 after reaching 79-90% confluency. At the end of treatment, cells were trypsinized and incubated with dye containing DPBS.

12/4/2015 5:40:30 AM  

Rodrigo Hoyos
Boston Children's Hospital

Thanks a lot for your answer, that's what i figured but I got confused since in step two it says "Cells were treated with NAC or H2O2 until 70-90% confluency reached" instead of after reaching 70-90% confluency.

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 Hsuan-Cheng Huang(1)