Hi Anusha,

Obviously the flow cytometry method is more reliable.

The reason why flow cytometry is the best way bcoz, it counts a constant number of cells and gives the fluorescence (example: 1000 units of fluorescence/10 000 cells).

But the plate reader reads the fluorescence/well which is critical as a lot of cells are being washed away when the plate is prepared for the assay. Since the treatment group supposed to induce more ROS than the control those cells are more prone to washed away and the plate reading looks wired because the fluorescence value would be higher in the control than the treatment group.

If you want to try plate reader make sure to normalize the number of cell/well to the fluorescence value you got.

Cheers..!

Susara

Hi Anusha,

Flow cytometry is more precise, however,

fluorescence based technique (fluorimetry using a multiplate reader) is much easier to perform and gives more amount of data. As same plate can be read at different time points. I have attached one of my article for your reference. Feel free to ask anything else.

Agreed, FACS is the more accurate of the two methods. However, chemiluminescence (using a chemiluminometer, luminol and the complete sample without washing) can be another option. It is also important to take into consideration the probes used as some are not equally sensitive to the different types of ROS and RNS, thus leading to differences in results.

Thank you Susara Madduma Hewage, Akash Sabarwal, Stefan S Du Plessis... The discussion was quite enlightening..

These papers show FACS based ROS measurement

https://www.researchgate.net/publication/283306685_Smac_Mimetic_with_TNF-_Targets_Pim-1_Isoforms_and_Reactive_Oxygen_Species_Production_to_Abrogate_Transformation_from_Blebbishields

https://www.researchgate.net/publication/299607409_Novel_PKC-_to_p47phox_interaction_is_necessary_for_transformation_from_blebbishields