~10% of the Reactive oxygen species can originate from extramitochondrial sites like NADPH oxidases. Besides knocking out mitochondrial genes, is there a way to determine the differences in cell culture. I am trying to design an experiment to explain that the origin of ROS is extra mitochondrial. As far as I know, fluorescence based assays do not tell the difference from where they come from. Am I missing something?
Hi,
you might want to use the H2DCF-DA probe for measurment of the ROS in cytoplasm together with mitochondria specific probe, which get sequestered in mitochondria and only measures ROS inside there...(like Mitotracker CMXH2Ros for instance)
hope this helps a bit.
Regards,
Zdenek
You may try Electron Transport Chain inhibitors to block the ROS generation from mitochondria, such as (chlorophenylhydrazone) CCCP, rotenone, and thenoyltrifluoroacetone (TTFA), etc.
Best wishes,
Kuiyi
@ Zdnek, Yeah got it, but if am using long incubations like 24 h or so, we still can't guarantee peroxide originated from mitochondria and passed to cytosol! About mito targeted probes, I think the incubation periods is short like half hr right?
@ Kuiyi, i considered those to use as check point if it can decrease ROS from mitochondria. However I have same question of how to apply these in long incubation periods. That to say, if you are looking for 24 h time, is it better to have these prior to measurement to completely block initial ROS release from mito?
I agree with other comments about MitoSOX. You should verify if your stimuli induces mROS. DCFDA is a good dye to detect cytoplasmatic ROS. Another idea is to block mROS with MitoTEMPO, a mitochondria-targeted antioxidant with superoxide scavenging properties. Be careful when using DPI and apocynin to block NOX and cytoplasmatic ROS. High concentrations of DPI (flavoenzymes inhibitor) may directly inhibit mitochondria activity. Apocynin (inhibits p47phox migration to the membrane) can indirectly inhibit mROS since NOX2 can induce mROS generation. Thus, you should verify if this kind of inhibitors are reducing mROS.
Good luck!
like everyone told you before me, Mitosox is a good tool. Please do not use DCFDA. It is not easy to measure production of oxidants using DCFDA as a probe. The number of controls and the method needed to have a correct interpretation are extremely difficult. You cant ever to Kalyanaraman, B., V. Darley-Usmar, K. J. Davies, P. A. Dennery, H. J. Forman, M. B. Grisham, G. E. Mann, K. Moore, L. J. Roberts, 2nd and H. Ischiropoulos (2012). "Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations." Free radical biology & medicine 52(1): 1-6. for better information on ROS probes. The problem with Mitosox is that it reacts with superoxide but not with hydrogen peroxide, and even when all hydrogen peroxide is formed from oxide, not all is produced from superoxide. So if you have mitochondrial hydrogen peroxide formation without changes superoxide formation, you will not see it. There are a few more selective probes for hydrogen peroxide, some of them targeted to the mitochondria that you could use simultaneously with a cytoplasmic hydrogen peroxide scavenger, such as overexpression of catalase that might work. The true is that there is not an easy answer to your question and the methods to use will greatly depend on the specific experimental model and conditions.
It is relatively easy to generate Rho0 cells or to buy them. These cells have no mitochondria and thus will act as a control.
You are a little harsh on H2-DCF-AM; it is a tool for investigation and as long as one is conservative and runs appropriate controls it is possible to get learge amounts of information from its us.