Hey everyone,
there are a lot of ROS measurement discussions and questions going on, so I wrote down our experiences of the last seven years "filled with a lot of ROS measurements".
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not produce ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would handle with care.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA, which a lot of people use. This also freely diffuses over membranes, so with that DCF probe you only detect "overall ROS" in the cell. Same goes for CellROX. DHE measures specifically superoxide, but also diffuses freely over membranes. With all these ROS probes you can investigate topics like "Are ROS involved at all in my setting?" or "Is oxidative stress increasing in my setting?". However, by only using one of these probes one cannot make a statement in which of the many compartments of a cell ROS are produced.
For some ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF) or are targeted to cellular compartments (MitoSOX). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2).
In my opinion, the assumption that ROS (especially H2O2, because of its membrane permeability), once prodcued, diffuse freely through the cell and somehow, somewhere hit their target without respect to the location and of the source of origin is wrong.
Cells established highly effective detoxification systems in each compartment (Cytosol, mitochondrial matrix). So only when ROS are produced in a uncontrolled and overhelming manner (because of mutations, diseases, some pathogens, cell death and so on) this could be the case. Many very good reviews clarify the difference between locally and controlled ROS production for specific functions (which can be turned off) and global destructive ROS production in harmful way.
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader. The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
Thanks Marc! Great overview and your paper has helped me a lot in understanding the mysterious ways of ROS.
Thank you Gillian :)
Please feel free to ask any time questions of any kind. I am happy to help. There is a fresh very good review about ROS. Unfortunately they did not cite us but that's life :D
Have a look at:
Sies, H., Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol (2020). https://doi.org/10.1038/s41580-020-0230-3
Marc Herb Thanks for sharing your experiences with ROS assay. I was also trying to measure ROS in Mtb infected THP-1 (24h post-infection) but didn't detect any ROS, probably the signals were lost post 24h. I read it somewhere that TNF-alpha can stimulate ROS in THP-1 after 3h. So what could be the ideal time to detect ROS in infected THP-1 post-stimulation with any drugs, according to your experiences.
Hey Mohd,
that is a difficult question :D First of all, I am not very experienced with THP-1 cells. In ex vivo macrophages, TNF does not induce ROS by itself, but influences ROS production after different stimuli (Hypoxia, infection, PAMPs).
As I wrote above, ex vivo macrophages (like peritoneal macrophages, microglia and so on) at least in our hands, react very quickly after chemical stimulation (PMA) or bacterial infection. Any type of immortalized cell line we used did not produce any detectable ROS levels with stimuli that cleary induce ROS in PMs, microlgia or BMDM. In RAW macrophages for example, we could not induce ROS production with any used probe (Isoluminol, Luminol, DCF, 6-Carboxy DCF, MitoSOX, DHE, Amplex Red) at least with the stimuli we normally use and which work in "normal" macrophages. Since THP-1 is immortalized, I am not sure, where to start.
I would try the following:
1) Use different MOIs for infection (for example 1, 10, 25, 50).
2) 24h might be to late, but immortalized cells might react slower. You can try 30 min, 1h, 3h and 5h post infection.
3) Use a positive control that triggeres ROS prodcution (for example 10 ng/ml PMA f.c. should do the job, please do not use H2O2).
4) You could use more cells.
5) Perhaps you are using the wrong ROS probe and miss the ROS production. Cells do not produce ROS anywhere and ROS do not flood randomly and equally the cell.
I hope that helps you. Please keep me up to date.
All the best and stay healthy,
Marc
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