Should cells be stained with an ROS dye before or after ROS induction?

What is the most common technique for staining cells with an ROS dye? Is it before or after ROS induction? For example, some ROS dyes such as Cat#22903 recommend staining after ROS induction. While other ROS dyes such as Cat#22901 recommend cell staining before ROS induction.

Does it make a difference if cells are stained before or after ROS induction?

As in the case of Cat#22901, the dye molecule is cell-permeable and non-fluorescent. Generation of a strong fluorescent signal only occurs when the dye reacts with ROS species. Therefore, cells may be loaded with Cat#22901 prior to ROS induction. This could prove useful in establishing a basal level of fluorescence indicative of natural ROS production. Upon ROS induction, the dye generates a strong fluorescence signal due to the increased levels of ROS.

Our scientist performed the experiment using both methods: stain then induce, or induce ROS first then stain. Both techniques worked, however staining as the first step showed a better response in our cell lines when using Cat#22901. 

It’s important to note that ROS is a natural byproduct resulting from the normal metabolic activity occurring in cells. Therefore, cells stained with an ROS dye will display relatively low levels of fluorescence intensity as it detects these natural ROS levels. When compared with corresponding ROS treated samples, the fluorescence intensity will increase significantly due to the high levels of ROS generated. Additionally, when performing ROS assays it is advantageous to begin with healthy cells as the major population in your sample. Unhealthy cells generate high levels of ROS, which can create background interference or a false signal