Hey Sheetal,

ROS measurements are done routinely in our lab (with macrophages however) but it took a long time to set it right.

First of all, phenol-red free medium is good, but we also got a lot of background and strange responses with either FBS or mouse serum. So for ROS measuremtents in a plate reader i would not recommend serum. We use HBSS and measure ROS for the maximum of 2 hours. But macrophages are quick to react, so after an our the ROS production goes back to normal.

MEFS (a non immune cell) produce ROS on a basal level but after LPS treatment or bacterial infection the signals dropped. This is because they are dying or shutting sown slowly. If your ROS levels drop below untreated cell levels this is either because the medium is quenching the signal or cells are becoming not happy.

I do not know your cell line but of course there is the possibility that these cells react with producing less ROS. For any information or techniqual detail for ROS measuremnts (with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 80% of it is more or less about ROS measurements in a plate reader.

https://stke.sciencemag.org/content/12/568/eaar5926

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

Please keep me informed, how it goes and ask any time for further help.

All the best and a nice weekend,

Marc

Hi Marc Herb,

Thank you for your suggestions. The cells I am working on are fibroblast-like synoviocytes.

Hey Sheetal,

so comparable to MEFS. TNF can also induce cell death. So your declining ROS signal might be because the cells are dying. I would recommend to try Phorbol 12-myristate 13-acetate (PMA) as a positiv control. We use it as a chemical positive control for ROS induction. It activates a lot in the cell very quickly, so you will get ROS production from a lot of different sources (NOX enzymes, mitochondria and so on), but when your cells do not produce ROS after PMA treatment, then perhaps the ROS probes are not sensitive enough for the low amounts of ROS your cell line generates. Not every cell reacts with an increase of ROS production to every stimulus. So leave your experimental setting as it is but treat also a set of cells with PMA. We add PMA in a small volume (10-25 µl) with a f.c. of 1 ng/ml right before the cells go into the plate reader.

Please keep me informed how it went.

All the best,

Marc

Hi Marc,

Thanks for your insights.

I use H2O2 as a positive control, and signals are quite intense. But definitely cell death might be a problem because I am getting more or less the same signal from treated as well as untreated cells.

Hi Sheetal,

people often use H2O2 as a "positve control" but I would rather not. If you give a substance in your well that you want to detect with your probe of course you will get a signal. But you want to know how and if your cells are producing ROS. It would be the same as if you would put IL-6 in your IL-6 ELISA and of course you will get a "IL-6-Signal".

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity". In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained). If you like, please have a look at: Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313

Please also have look at our Perspective article, if you like:

Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...