Total ROS measurement protocol - (Oct/19/2009 )
I work with human lung carcinoma a549 cell line and I want to measure total ROS production as a result of treatment with certain drugs. Do you any protocol I could use?
I should mention that I would prefer to use a protocol in which the measurement can be done through optical absorption and not fluorecence.
Thank u very very much!
Hi! You can use the Nitro Blue Tetrazolium method which measure the total superoxide production. This method consists in the incubation of your cells with treatments and 0.2% of NBT. After the incubation, cells are centrifuged (3200g, 2min 4oc). Discharge the supernatant and add 100uL 50% of glacial acetic acid. Use the sonicator to lysis the cell (3 pulses of 6 seconds) and read it at 560nm.
In this process NBT reacts with superoxides and forms formazan. There are a lot of articles using this method as measurement of ROS, but particularly I think it's important to discriminate which specie you are measuring.
For a total ROS production the most indicated method is using the DFC fluorescent dye which reacts with all kinds of ROS.