Hi Ara Ro! A simple and rapid method for oxidative stress measurement would be by DCFDA fluorescence measurement by fluorescence microscopy or markers of lipid peroxidation as TBARS by ELISA or HPLC.
Hi as suggested by Mauricio, DCFDA based detection of ROS would be reliable method for determination of oxidative stress invitro. However there are enormous literature based on free radical scavenging properties. However those measurement would not have any impact on intracellular antioxidant potential.
Even with DCFDA based intracellular ROS measurement, a right oxidative trigger is essential to determine the redox property of material.
Most commonly used oxidative triggeres are H2O2 or oxy-LDL.
Invivo, measurements include TBARS or ABTS, but these measurements also measure free radical scavenging property.
It would be great if you could clarify on your question.
Hi Ara,
Your question have been asked by Roksana and extensively answered earlier by me and other experts in this area. I totally agree with Mauricio and Paul that DCFDA is the best for such purposes and I also had good luck with this probe.
You can read further following the older thread
https://www.researchgate.net/post/What_are_good_fluorescent_probes_for_measuring_general_oxidative_stress_in_cells?ev=tp_feed_post_xview
We should create a thread in the RG forum regarding the numbers of similar questions asked and answered by experts in respective areas!!!
Best-
Hi Ara,
DCFDA is certainly the standard in the field. However, if you are willing to go an alternative route, I suggest trying Promega's ROS-Glo. While I haven't tried this particular assay yet, I have had excellent success with all of Promega's luminescent-based assays in the past. These assays typically give you a great dynamic range and are very reproducible. And, if you are looking for a straight-forward assay, it does not get any easier than this. If you have a luminometer compatible with 96-well plates, it might be worth giving ROS-Glo a try.
Best,
Nate
Ara, Promega's ROS-Glo is extremely simple. All you have to do is:
1) Add the ROS detection solution.
2) Incubate at room temperature for 20 minutes.
3) Measure luminescense.
Protocol is here:http://www.promega.com/~/media/Files/Resources/ProtCards/ROS%20Glo%20H2O2%20Assay%20Quick%20Protocol.pdf
as mentioned by the others, DCFDA is certainly a way to measure ROS. ONe pro to this approach is it can be used to measure ROS in live cell. It is however nonspecific. If you are interested in measuring specific types of ROS such as mitochondrial superoxide, consider using MitoSOX (which can also be used in live cell imaging). Amplex Red can be used to measure extracellular hydrogen peroxide formation. Finally, thiol modication reagents such as Bodiy IA or BIAM or biotin switch assays can be utilized to measure protein thiol oxidation.
Hope this helps.
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