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How to estimate the induction of ROS in cell culture?

The answer depends on which kind of probe do you use for a detection of ROS - superoxide or h2o2 specific probes. And another concern is in which cell compartment do you want to induce the ROS production - cytoplasm, mitochondria, peroxisomes. If you are using DCFDA (personally i try to stay away from this probe due to its artifacts which is very difficult to correct for) you can use any inducers of any kind of ros which release them into cytoplasms. Or optionally you can decrease the antioxidant defense by Tetr-butyl-hydroperoxide or ethacrynic acid. If you use DHE (superoxide specific cytoplasmic probe) you need to generate superoxide in cytoplasm to get a positive control because this probe will not detect the h2o2. Also depending on the cell type you can try to activate the NOX or other plasma membrane associated sources, inhibit the complexIII by antimycin A (that will produce some superoxide (50%) in the cytoplasm ) or menadione. The latter from my experience work differently in different cell types and i suspect its major effect on induction of ROS is through depletion of glutathione pool. For positive control in mitochondria again you need to check with the probe, if you use mitosox the straightforward positive controls are rotenone and antimycin. However some cells do not response on rotenone but all which i tried responded on antimycin A. For mitochondrially targeted hyper or redoxGFP all respiratory chain inhibitors are fine as all superoxide will be converted to h2O2. One more comment on mitoSOX which i am working on and had to optimize and correct. MitoSOX is mito membrane potential sensitive and therefore one should always track the changes in mito membrane potential when uses this probe. The best way to do it is to normalize for TMRM fluorescence as both probes accumulate similarly in cells (the incubation time is min 1 hr, better 2 hr). Another issue with mitoSOX is recommended concentration. 5 uM is too much for mitos because of the TPP moiety of this probe which is toxic above certain concentration. I use 0.5 uM in BSA free medium. People use 1 uM-2 uM in 0.3 % BSA if cells can't survive without BSA. And the third problem of mitoSOX is unspecific oxidation in the cells using recommended wavelength settings. (There are plenty of literature on this topic if someone wants to do it correctly). So we use different setting for its detection - 390 excitation, 617 emission which is specific for superoxide oxidation (also published in 2006 or so). There is no ideal probe all need some corrections before interpreting the results. I am happy to help more with Hyper and mitoSOX troubleshooting which i use widely.


Souce: NovoPro    2018-05-08