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Total ROS measurement protocol

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#1 olga_m



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Posted 19 October 2009 - 12:48 PM

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!

#2 hillken



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Posted 23 October 2009 - 07:12 AM

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.

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