Is there any protocol to measure intracellular ROS using spectrophotometric method?
ROS can hardly be measured directly by spectrophotometry. Maybe measurement of stable products of free redical oxidation is the right choice. I don't think that it's real to measure all oxidated products; instead it's better to select few robust indices, such as level of TBA-RS or G-SH.
Few times I've seen how researchers tried to introduce some complex indices to assess the ratio of ROS generation / removal, but all these values are based on integration of few separate measures (organic peroxides, TBA-RS, SOD, GPX etc.).
You may try to use DHE or DHF in the dark then run fluorometry but not spectro.
search for DCFDA in abcam its a dye based protocol and compatible with flowcytometry and even spectrophotometer.
Thanks for your consideration, i want a method with out using kit, is there any protocol for measurement?
I would suggest H2DCFDA as a ore global assay for ROS. We use this dye in vitro and its stable enough for FACS as well as fluorescence measurement.
Our protocoll: use 5-10µM H2DCFDA and stain the cells in dark for 20-30min at 37°C. Wash the cells twice with PBS and measure your flourescent signal. If you want to study kinetics of ROS formation, you should use medium without any colour, e.g. phenolred-free RPMI.
If you want to check for a certain oxygen species (superoxide, hydrogen peroxide) to make a clearer statement, you could also use AmplexRed, luminol, luceginin etc.
Best
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioecology
- Systems Ecology
- Landscape Ecology
- Connectivity