When my cells are exposed to a drug, they present reduced cellular ROS. So, I'd like to compare my results with standard NAC treatment. In your experience, which is the optimal concentration? For how long a time?
I will test concentrations ranging between 1-10mM (usual in articles). After that I will post here my results.
a common concentration is 10mM-which is rahter high-you should do titration- when you dissolve NAC, it´s becoming acid- so, adjust the pH. some cells tolerate NAC for more than 24h....you can also comparewith glutathione-treatment...or BHT (which is an aromatic antioxidant and might activate the xenobiotic-pathways (NRF2...), this drug is also not tolerated by all cell-types...
Actually it was a comment from my paper reviewer (use of NAC). So, I must do it. I will be aware about pH! Thank you for your tips...certainly it will be very useful!
Ulrike Resch, recently I've tested tert-butylhydroquinone (tBHQ) in my culture. It was not bad.
I agree with the suggestions made so far but like to add something. I think it is worth the effort to monitor the actual GSH/GSSG situation in your cells. I would do this in all conditions used, i.e. with/without NAC treatment and each additional treatment you might apply.
In my experience the GSH/GSSG content of the "same" cells might differ, e.g. from day to day or passagewhise. The resulting problem might be that you don't detect an antioxidant effect with NAC due to an already very high GSH-concentration the cells.
So, to sum it up: check your GSH/GSSG levels! I suggest the Glutathione-Reductase-Recyling-Assay using the detecting of DTNB in a platereader.
Antonio, I am not able to answer your questions yet, however it will be certainly needed! In my first two tests NAC could reduce ROS, but, unexpectedly cell viability was poor. I am testing again just now. I deeply thank you all for your tips!
Hmm, did you check for pH in your NAC Stock and/or monitor the pH-Change in culture medium after addtion of NAC? Might be a reason fpr poor viability...
Yes, I measured and adjusted it. Also, I re-filtered the medium before using. It was totally unexpected. Today I put my cells in culture with NAC again. And amplified the range of concentration ... I will check after overnight incubation. Do you often use NAC, Peter?
A quick question to this discussion if anyone is around. Does one have to re-feed NAC after a certain couple of hours for a 72 hr long cell culture experiment? Or does 20mM NAC suffice for that long? I have been seeing a decrease in ROS generated by hydrogen peroxide at 24 hrs following NAC treatment, but this effect fails to show up at 48hrs and longer. Hence do we know the stability of NAC in cell culture?
Hi, @Luiz Fernando Quintanilha, do you have an optimal concentration for NAC Treatment now? I use 5mM to pretreat the Hela cells for 24h or 1h, it seems that NAC didn't work as an antioxidant but lead to higher ROS Signal and lower cell viability. The pH value is around 7.2 after the addition of NAC. Do you have some suggestions? Thank you very much
Hi Fangxia Shen, how are you?
Actually I could not keep doing this research, so I cant contribute much from now. Good Luck
@fangxia Shen: We have experienced similar issues with prolonged use of NAC. It increases ROS instead of suppressing. I believe a better way to come around the ROS is to use either Vitamin C or Vitamin E, which are natural antioxidants and they provide better response. At least in my setting these two worked better than NAC. I am still looking for the explanations as to why NAC at prolonged incubation fails to quench ROS.
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