Might I add one or two additional angles to the discussion?

1) what about checking to effect of NAC in terms of GSH/GSSG measurements?

I learned "the hard way" that not every fibroblast on every day reacts in the same way to NAC treatment. Key Questions: How (quantitatively) does NAC change the actual total GSH AND GSH/GSSG ratio in your cells in each specific experiment?

2) time might be an interesting factor

Check the reaction of your cells (both as your orginial concept and as described in 1) over time. I would suggest trying 2h, 4h, 6h, 24h, 48h. Key question: How do your cells react to the NAC treatment.

In summary, I think more control & data on the effects/kinetics of the NAC treatment might benefit your experiments! Do assume what NAC does - check it! ;-)

Hi Gail,

You have answered your question. I worked with NAC before and had the same problem that you mention. The variation of pH due to NAC produced cytotoxic effects on my cells. As you pointed out, the only way I found to quenching this alteration was by readjusting the pH media to normal and then add it to the cells.

Best,

Julio

I think NaOH is appropriate for adjusting the pH. I'm assuming you are using a balanced salt cell culture media like DMEM or similar? If you think the pH is causing the elevated ROS, you test this hypothesis by lowering the pH of the media without NAC and see if the ROS is still elevated. Either way, you would want the pH to be the same in test condition as control.

Hi Jason,

we've been working with NAC and human fibroblasts for several years, please read the manuscripts regarding X-linked adrenoleukodistrophy (X-ALD)human fibroblasts (papers of "Pujol A" group members).

Thanks to you both for answering so quickly!

Jason, I should have mentioned that I am using DMEM, thanks for your advice on checking my theory about the pH!

Julio, thanks for clarifying, I have met nobody here who has experience using it and most papers don't mention about adjusting the pH of the medium.

Thanks again for your help, I really appreciate it and I am sure my poor cells will too!

Thanks Francisco, I will read the paper you have recommended!

Gail, you can either adjust pH using NaOH or increase buffer capacity in culture medium. In order to increase buffer capacity you may also let cells to rest in the incubator for 1-2 h at 5% CO2 or at last use L-Glutathione-Reduced instead of NAC. Good luck!

Daer Gail,

Always adjust the pH to the value that your cells are growing in. We used NAC on a number of cell lines, and with adjusted pH, no deleterious effects were noticible

of course NAC is a derivative of cysteine which is slightly acidic amino acid (weak acid) so it can change the pH value so you must adjust the pH value to the value that your cell line is growing in and this by using steralized NaOH solution

Your discussions remind me of something

NAC can completely inhibit oridonin induced apoptosis in cancer cells. It is known that oridonin can increase the ROS level in cells. So, NAC may not inhibit the ROS induced by oridonin, but directly bind and inactive oridonin.

In my hand after adjusting pH, NAC worked fine in primary culture, however, I have seen that the concentration was critical to maintain low ROS level depending on the agents used to induce ROS. We need to make sure that whether it reflects the true nature of ROS induction from variety of organelles so the source of ROS is crucial to see the end effects of NAC. MitoROS was indeed inhibited by NAC as did for ER stress.

Thanks so much for all of your replies! It is always good to get other perspectives on experiments and I really appreciate that you have taken the time to try and help!

I am working on human fibroblasts expressing fusion oncoproteins (which are inducing elevated ROS) and I am culturing them in 3% oxygen. I am attempting to block induction of ROS to determine whether it is a necessary step in induction of senescence by these proteins.

I have adjusted pH and am repeating the pilot experiment with varying concentrations of NAC. Thanks for the info on NAA! I wasn't aware of that!

I will keep you up to date on how it all goes....hopefully well...

Thanks again,

Gail

NAC is a source of cysteine, and as such it may do many different things in cells. At high concentrations, you can be altering the sources of aminoacids for cell metabolism.

It can produce many signalling effects, as Cys is in the pathways for methylation, H2S,...

Any redox effect should be tested and not assumed. For example, it can help to maintain or increase glutathione levels, but this effect would be more evident when there is a glutathione deficit, which may not be the normal situation in cell culture.

Hi Gail,

And what did you use for ROS detection? NAC can show complex effect depending on cell type. Did you check pH in the NAC solution that you added to the cells? You can buffer this solution if you have real pH shift.

Hey,

I have just been using DCF-DA detection of ROS so it is fairly non specific.

I have checked the pH and adjusted it which appears to have helped with the elevated ROS in treated samples, however, I am not sure that I am seeing any really difference in ROS levels in different doses.

Thanks again for all answers!

I have used 1 mM preincubation of NAC 2 hrs and this protects against ROS induction by 200 uM Cd acetate (which is a high amount of Cd).

Probably you can try 1 mM NAC and 2-3 hrs preincubation.

Might I add one or two additional angles to the discussion?

1) what about checking to effect of NAC in terms of GSH/GSSG measurements?

I learned "the hard way" that not every fibroblast on every day reacts in the same way to NAC treatment. Key Questions: How (quantitatively) does NAC change the actual total GSH AND GSH/GSSG ratio in your cells in each specific experiment?

2) time might be an interesting factor

Check the reaction of your cells (both as your orginial concept and as described in 1) over time. I would suggest trying 2h, 4h, 6h, 24h, 48h. Key question: How do your cells react to the NAC treatment.

In summary, I think more control & data on the effects/kinetics of the NAC treatment might benefit your experiments! Do assume what NAC does - check it! ;-)

Gail, if you add NAC to medium and the color of phenol red in medium stays the same, your pH is ok. I'd suggest something similar to Faraz and Peters thoughts, which is to use 1-5mM NAC, incubate for 2-4hrs, then run glutathione assay (Tietze recycling assay or others) to convince yourself that total glutathione and preferably GSH/GSSG ratio is increased. Importantly, make sure that GSH is increased "enough" to counter the oxidative stress of your treatment.

You now have established that your pre-treatment setup is valid and can do NAC pre-treatment, wash, then post-treatment of your choice without people questioning whether NAC is directly reacting with your treatment.

Thanks guys, these answers have been great and really helpful! I have managed to buffer the NAC so seem to have resolved the pH issues!

I will look into the glutathione assays and think carefully about my controls!

Thanks again, really pleased with the responses I have received and glad to see that the scientific community is so willing to help out those with less experience!

Gail

I agree, the most important thing is to buffer NAC solution to 7.2

I don't know if that was said but you could also try a lower range of concentrations.

I personally use 10µM of NAC to induce drop of ROS generation (around 1.5 fold decrease compare to stressed conditions)

My experience is that many thiols show a biphasic ROS response, at low concentrations their auto-oxidation can generate more ROS than they scavenge. This occurs particularly if transition metals or similar components that can catalyze their auto-oxidation are present in the media. At higher concentrations they scavenge more than they generate and diminish ROS levels. I tend to prefer using thioglycerol over NAC as its neutral (so no pH problems) its super-soluble, its readily cell permeable, doesn't generate cysteine and for most cell is very non-toxic.

NAC is a direct-acting antioxidant. Nrf2 is the transcription associated with cellular endogenous defences. Nrf2 is activated by a range of stressors and many of these are mild pro-oxidants which are sufficiently pro-oxidant to activate Nrf2 but not sufficient to alter the cellular redox milieu. When you add NAC, you will inhibit Nrf2 and therefore inhibit the gene expression of cytoprotective genes such as those coding for GSH, SOD and a range of other endogenous antioxidants. As a result, you are likely to see an increase in ROS.