I have luminol and a spectrophotometer (and basic lab equipments and instruments), nothing more. My treatment (Hydrogen peroxide) will cause production of ROS inside the HEK293 cells and I would need to be able to measure this. My specific questions:
a) Is it possible to make a standard curve for ROS concentration using Luminol? How?
b) Could you point me to papers that might have done this before?
c) Is the concentration (in mM, say) of ROS actually measurable (directly or indirectly)?
Luminol can be measured by chemiluminesence and gives both extracellular and intracellular ROS. Lucigenin is another probe.
Similarly you can try to use DHE and DCF but these are sepcific for either superoxide or hydrogen peroxide, whilst Luminol will measure all ROS.
The way in which you will have to quantify it mught be challenging. Perhaps express as per number of cells (e.g. relative light units per million cells or absorbance per number of cells.
Hello ,
Both of you have given me inputs I could work with, but I now realize that I should have stated one other problem that I'm currently facing :
How do I construct a standard curve for ROS using H2O2 in luminol? Any idea in this direction would be appreciated. Even if there is a method which uses DCFDA, it is fine, as that is a chemical which I could buy.
First, you need to decide what type of ROS you want to quantify and where in the cell, in the cytosol or mitochondria. The closet method to specific in quantifying superoxide, in my opinion, is HPLC.
Incidentally, DCF is definitely not specific for H2O2. I don't know why people keep saying this
Hi Shashank,
Instead of creating a challenging std curve for the experiment, why don't you just put the control as 1(normalization by dividing the absorbance of control with other all groups in an experiment) in DCFDA assay..after subtracting blank from all groups..assuming that you are checking other experimental conditions against a control group.
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