MarkerGene™ Live Cell Fluorescent Reactive Oxygen Species Detection Kit
Product ID: M1049
This kit provides all necessary reagents, buffers and a detailed protocol for the detection of Reactive Oxygen Species (ROS, peroxidase activity) in live cells.
In healthy aerobic cells, ROS generation occurs at a controlled rate but under high stress conditions, its production is greatly increased, resulting in changes of many cell components including proteins and lipids.
Fluorogenic peroxidase substrates are converted to highly fluorescent products in the presence of the enzyme and hydrogen peroxide but can be relatively unstable for use in enzyme-linked immunosorbent assays (ELISAs). By forming the diacetate derivates, intracellular applications are more efficient, whereupon the acetates are cleaved by endogenous esterases, releasing the intact substrate. In the presence of nonspecific ROS, commonly produced during oxidative stress, the fluorescein substrate becomes oxidized, emitting green fluorescence.
The MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit utilizes a cell permeable substrate that is a reliable fluorogenic marker for ROS detection. Upon enzyme activity, a highly fluorescent dye is produced, with EX: 495nm and EM: 530 nm. In addition, this kit also contains t-butyl hydroperoxide, a common ROS inducer, as a positive control.
The assay kit contains enough reagents to prepare up to 250 mL of staining solution, or approximately 100 assays (12-well tissue culture plate format), or 100 x 96-well tissue culture microtiterplate staining assays. Methods for testing various drug or inducer compounds are also described. This assay can be adapted for high-throughput, high-content assay screening analyses.
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Please note, it is not necessary to fix the cells prior to loading this substrate, as it will passively diffuse into live cells. Nevertheless, I don't think that the fixation step should significantly affect the activity of ROS enzymes. But the substrate and the product may become more permeable and show some diffusion (bleeding) from the fixed cells, more than with live cells. If this is the case, you might have to monitor staining and either lower the concentration of the substrate or adjust the staining time (shorter time) to modulate the possible diffusion effects.
If you are counterstaining with an antibody, it might be better to stain first with antibody, then stain with the ROS kit.
Either method is fine, but it depends on your application. But whichever method you choose, you have to keep that same protocol for all assays, so that they can then be compared.