Recently, I am performing the intracellular ROS detection by DCF-DA method in SHSY5Y cells through flow cytometer. But, I found a high fluorescence intensity for the untreated control stain with the probe DCF-DA at 10 micro molar for 30 min, 37 degree celcius in dark than that of the ros inducer i.e. rotenone. Why is it happening like this? Please tell me.
Hey Hijam,
your staining protocol for DCF sounds good to me.
As far as I know (and what we see in macrophages), rotenone induces ROS production only into the mitochondrial matrix. It blocks the binding site for Ubichinon at complex I. Since the electrons, which are delivered by NADH, cannot be transfered to complex III, complex I becomes fully reduced and electrons flow back and accumulate in the mitochondrial matrix to form ROS. Rotenone is a commonly used as a positive control for the ROS probe MitoSOX, which measures ROS inside the mitochondrial matrix. DCF, however, measures ROS mainly in the cytosol. We observed that cytosolic ROS production was strongly reduced in rotenone-treated macrophages. Of course it can be completely different in your cellular system but rotenone is not "just a ROS inducer" in cells. You have to overcome the very effective antioxidant defense system of the mitochondrial matrix and then the ROS have to bypass to membranes. So it has to be a very unhealthy massive overproduction inside of the mitochondria to "induce ROS" in the whole cell. It also highly depends on the cellular location where you are measuring ROS production. For some examples how ROS measurements look in different compartments (with rotenone) you can have a look at our paper. How and where macrohages produce ROS is one main topic of this manuscript.
Herb M, Gluschko A, Wiegmann K, Farid A, Wolf A, Utermöhlen O, Krut O,
Krönke M , Schramm M. Mitochondrial reactive oxygen species license pro-inflammatory signaling through disulfide linkage of NEMO. Science Signaling. 2019 Feb
Feel free to ask any further question.
All the best and a happy new year 2020,
Marc
Thank you Marc.
Sure, i will read your paper and ask you if i have any problems.
Happy New Year 2020 to you too
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence