If you want to save money you can skip subgroup 1. It is enough to get interesting results in subgroups 2 and 3 to prove your point.

But I have to make a question. Why are doing this experiment?

ROS will be increased. This we know without doing any further experiments because it has been tested in the past.

Thanks for answering. Yes, we know that Doxorubicin does increase ROS.

I have shown that redox enzymes are regulated by drug cocktail. I am doing this experiment for the validation of ROS regulation by redox enzymes.

Quantification of ROS and RNS production is a good idea.

Perhaps you would be also interested by measuring the level of oxidative damages, such as products of glycoxydation, lipid peroxydation or protein carbonylation. It would be another way to assess the efficiency of antioxydant enzymes.

Thanks for the advice Janek!

Dear Kim,

I have some experience on working with cancer cells. My first and most important question, when you culture your cells , do you use antibiotics? Most people use PenStrep. Then we can proceed further.

Alexander Panov

Hi Dr. Panov.

Yes, I do as well. Does it affect ROS generation?

Instead of an answer I have attached my paper on prostate cancer cells mitochondria. It looks that during the last 20 years I was the only one who isolated mitochondria from cancer cells grown without the presence of antibiotics. All antibiotics similar to streptomycin and gentomycin are mitotoxic.

Thanks! I will look into it.

Best,

Bumjun

positive/ negative control could be an asset.. finding a proper ROS scavenger (better not NAC) may give you the lower limit, while KBrO3 or other oxidizer may give you the upper one. By and large all the above assays are not accurate. HPLC with proper internal standards would be the perfect approach.

Hi Dr. Lyakhovich. Thanks for the valuable answer.

I saw that you published a number of papers with ROS measurements, mitoSox and DCF-DA were predominantly used for your research.

I was going to use DCF-DA since it is the most popular and easiest dye to use for intracellular ROS measurement. However, the more I look into this field, it seemed to be considered as a unreliable dye for ROS measurement by leading experties due to several reasons like indirect interaction with ROS species and artifactual amplification of fluorescence by redox-cycling of dye.

As you have mentioned, HPLC would be the most accurate method for ROS detection, but I need to do it in a short period of time. I am not sure I have enough time to optimize experimental condition of HPLC measurement. Thus I excluded HPLC method and chose above dyes which are relatively reliable compared to DCF-DA or DHR.

I guess it all depends on what your focus is and where you submit the paper, but from your experiences, were reviewers not being skeptical of using DCF-DA dye for ROS measurement in cancer?

Dr. Kim,

Thank you for paying attention to my works, - you probably saw that I am also in that queue of DCF-DA-users because it is easy and less expensive. But I acknowledge all drawbacks too. Perhaps, doing FACS with DCF-DA exposed/ washed cells +/- antioxidant(s) may provide you comparative information. In addition, probing the blots of whole cell lysates for carbonylation or similar ROS-mediated derivatives would be a plus. Kits with specific antibodies are available.

Kind regards,

Alex

Thank you so much for your kind answer.

I will look into carbonylation kit as well.

Have a wonderful weekend,

Best,

Bumjun

Hello!

I am experienced in measuring H2O2 specifically and in real time from single human and murine monocytes, using electrochemistry. In principle, this method is applyable to all single cells and to suspensions of cells. (see recent publication in ARS: Bozem, M. et al. - attached). We also determine H2O2 extracellularly with Amplex UltraRed. It is quite reliable, as confirmed by electrochemical and ESR measurements (also in the paper cited). Never use Amplex in medium, but in PBS buffer instead. I would also not recommend DCF-DA because of many reasons.

Please contact me, if you need further info. Monika Bozem

Interesting paper. Although some labs have sEM, I trust the question was about ROS rather than H2O2. Even if you subtract degraded H2O2 (assuming it will result in some ROS), the kinetic will not tell you much regarding ROS amount.