Hey Camilla,

as far as I know is MitoSOX not fixable and looses its fluorescence once fixed. But you can measure 12 cell lines at a time in a black 96er well plate in technical triplicates + rotenone as a postive control. Then you could test all of your 30 cell lines on two plates (of course one after another). If you like, I send you over the protocol for MitoSOX ROS measurements in a plate reader and a plate scheme. We measured ROS with MEFS and it worked quite well.

All the best and stay healthy,

Marc

Dear Camilla!

You also look at this article:

Article Mitochondrial Dysfunction Accounts for the Stochastic Hetero...

Cells.

MRC-5 human embryonic lung fibroblasts were from ECACC and P43, and P100 fibroblasts were obtained from adult skin

ROS levels, mitochondrial membrane potential, and mitochondrial mass.

To measure mitochondrial superoxide, cells were stained in 5 μM MitoSOX Red (Molecular Probes, http://www.invitrogen.com) for 10 min at 37 °C, and FL3 median fluorescence intensity was measured by flow cytometry (Partec PAS, http://www.partec.com). Specificity of MitoSOX for superoxide has been shown by the manufacturer, and its mitochondrial localisation was tested by co-staining with Mitotracker Green (unpublished data). Cellular peroxide levels were assessed by staining with 30 μM DHR (Molecular Probes) for 30 min at 37 °C and analysis of FL1 fluorescence. As a positive control, cells were treated with H2O2 (0 to 400 μM for 30 min) before DHR staining (unpublished data). MMP was measured as the FL3/FL1 ratio after staining cells with 1-μg/ml JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes) in phenol-free RPMI 1640 (Sigma) for 30 min at 37 °C. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (20 μM for 30 min) was used as positive control, resulting in a 60% decrease of the JC1 ratio. Mitochondrial mass was measured as FL1 fluorescence after staining of cells with 10 μM nonyl acridine orange (NAO; Molecular Probes) for 10 min at 37 °C in the dark. The flow cytometer was calibrated using fluorescent microspheres. All data are mean ± s.e.m. from n independent experiments with measurements in duplicate and 104 cells per measurement.

Cells growing on coverslips were stained as above and observed using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, http://www.zeiss.com). Conventional transmission electron microscopy was used to confirm NAO staining. Twelve sections of cells with central nuclei per group were systematically random sampled to measure the volume density of mitochondria per cell.

Marc Herb Thank you for your answer. Please sent me the protocol, that would be very helpful, thank you so much.

I used flow cytometry to measure the MitoSOX assay and it worked quite well with tumor cells. Maybe you can also consider flow cytometry to measure several probes in parallel.

This is also a good approach mentioned from Nadine Wiesmann. MitSOX works very well in Flowcytometry (as long as the cells are not fixed).

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...