Hello Abdullah Al Tarique

Yes, it is possible.

1. Cells may be cultured in standard culture media in multi-well plates (approximately 25,000–30,000 cells per well).

2. The medium should be changed the day before the experiment.

3. Cells should reach 80–90% of confluence on the day of the experiment.

Please note: You need to adjust the number of seeded cells and the culture duration to suit the specific cell type you use. Avoid using phenol red (background fluorescence).

4. Treat the cells with the drug at the indicated concentration for an appropriate time.

5. Then wash the cells twice with PBS to remove the medium and subsequently incubate for 10 min (needed to allow the probe to enter the cell and start the reaction within the mitochondria) at 37 degree C in 0.5 ml of measurement buffer containing 5uM MitoSOX Red.

Preparation of MitoSOX Red:

MitoSOX Red (on the day of the experiment, a working solution is prepared from 5 mM DMSO stock (50 mg in 13 ml of DMSO), diluted in measurement buffer). Protect from light.

6. After the incubation, the cells should be washed twice with PBS. The fluorescence can be monitored in the measurement buffer with a microplate reader set to 510 nm excitation (Ex bandwidth: 10 nm) and 595 nm emission (Em bandwidth: 35 nm) wavelengths.

7. Alternatively, if the signal is high enough, the fluorescence can also be measured using 400 nm excitation to measure only the superoxide-specific product of MitoSOX Red oxidation. This is a single-read measurement that does not define the kinetics of the reaction.

You may want to refer to the article attached below. (See Procedure, section 2.3.1.2).

http://sm.unife.it/it/ricerca-e-terza-missione/ricerca-1/ambiti/signaltransductionlab/publications/143.pdf

Best.

The mitoSOX assay, I believe measures superoxide (O2.-) which is a relatively innocuous compound itself, but it can give rise to far more toxic species. see this ref

Article Erythrocuprein, also Known as Superoxide Dismutase, Is a Hyd...

There are several chromophoric assays for O2.- , but it may be wise to also assay for the products of ROS mediated damage e.g., 8-oxoguanine, malondialdehyde etc.

Good luck.