I would like to measure ROS levels (total-ROS, superoxide and peroxides) in cell-free follicular fluid samples by spectrofluorometer. Could anybody suggest me some fluorescent probes or provide with a successful protocol? Thanks in advance!
Kindly check www.ncbi.nlm.nih.gov/PMC/article/PMC3165889. Best wishes
Dear Gabor.
The hydrogen peroxide is easily measured with Amplex red method.
Superoxide radical is much more difficult to measure in solution. Though in the incubation medium with mitochondria SOR is measured by production of H2O2. This is possible because mitochondria possess highly active superoxide dismutases 2 and 1 (in the intermembrane space).
There are however two questions: 1) What is the source of water you use? The Millique water or other system, which uses resins to remove ions, is usually highly contaminated with peroxides; 2) What is the purpose of your measurements?
In addition to the quality of water itself (basically you have to use double distilled water), some spectrofluorometers, for example Perkin-Elmer, are not equipped with devices for burning ozone, which is produced by the power lamp. Therefore, often the solution, if kept open, begins to accumulate peroxides at high rate. I have experienced this a couple of times, while working in someone’s labs.
And also, what is the purpose of your measurements from the scientific point of view? What conditions, objects, etc. Without knowing this, it is difficult to give any advice.
Good luck.
Alexander Panov.
Dear Professor Panov,
We would like to measure pure human follicular fluids after centrifugation (without any treatment). So this is not a culture medium, however it basically contains carbohydrates, proteins, trace element (selenium, cadmium, lead, zinc, copper and iron).
Unfortunately, I cannot give answer to the type of distillation.
Thank you for your help!
Best regards,
Gábor Máté
There are options. You want to avoid dyes that require esterase cleavage (dyes with AM esters or diacetates).
However, you can try using dihydroethidium (DHE), CellROX Orange, or CellROX Deep Red (but not CellROX Green unless you add DNA to the solution). None of those require esterase cleavage. I also agree with Alexander's recommendation of Amplex Red (also there is Amplex Ultrared, which gives higher dynamic range).
The paper that Charles recommended looks good, but be aware it requires a luminescence readout with a luminometer, which can be more sensitive, but also very short-lived signal.
Amplex Red in the presence or HRP is both sensitive and specific for H2O2.
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