Dear scientific fighters,
Has anyone met this problem?
NAC is supposed to be ROS scavenger. At beginning it worked fine, but later in my experiment, NAC increased ROS. pH is neutral. I used 20mM NAC, Bon-1 cell line, incubation for 2h. I did dose effect before, 20mM is better for this cell line. But now it is not working. Help..
Sounds like stability issue. Have you tried to troubleshoot this? Prepare a fresh solution of NAC and try again. How do you store it BTW?
I only used TEMPOL, stored at -20deg C. It worked well.
@Tilen Kranjc Thank you..and yes i tried to prepare stock again but still not working. i dissolved NAC in ddH2O and stock is 100mM. but stored at 4 degree.. i wanted to try again using minipore water instead of ddH2O and re-prepared all the agents including NaOH which i used to adjust pH..also when i incubate cells with NAC, which one do you suggest PBS or medium without phenol?
@Ge Yan First, I would keep the cells in full growth medium also during the treatment with ROS scavenger. I would just add the NAS to the growth medium, incubate for 2 more hours and then change the medium for the desired assay. If you need to change medium before treatment (for example if you are doing live imaging) then you should use phenol free medium. I was using PBS with HeLa cells, which are quite robust, and they typically started dying after 30 minutes.
Another thing to consider is the medium you are using. DMEM typically contains Na-pyruvate, which is a ROS scavenger itself and is therefore not suitable for such experiments. You would need to get DMEM without pyruvate.
TEMPOL is another ROS scavenger. I was considering NAS or TEMPOL and then decided to go with TEMPOL. I used it to rescue the effects of H2O2.
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