I want to measure nitric oxide released by huvec cells following treatment with chemicals. Can I use the Griess test? I read on RG that Griess test is not the best for human cells. In that case what would be the modified Griess test applicable for human cell lines? Also, can you express eNOS in HEK293T cells and measure NO release following treatment?
Griess isn't very sensitive. You could do ROS, use a kit (abcam ab65328), or DAF
If you overexpress eNOS in HEK293T, even Griess reagent should work. But Griess is one of the least sensitive method.
Actually, on of the most successful indicators for nitric oxide has been 4,5-diaminofluorescein diacetate (DAF-2 diacetate) (Chem Pharm Bull 46, 373 (1998); Anal Chem 70, 2446 (1998)).
I have to say that Griess is the least sensitive. Following are some suggestions...
DAF2-DA (or even any of the recent more-sensitive modification of this probe) is really a good choice. However experimental conditions determine the successful use of this probe. Assuming that you will be imaging intracellular fluorescence by microscopy, use sub maximal concentrations of a drug to stimulate NO generation. You can use some nonspecific agents too to see the maximum fluorescence/NO release. When your monolayers of cells are ready for experimentation, you will have to serum-starve them before treating with something for NO release. Here comes the frustration, cells tend to get detached or lose viability, if not during this step, later on during the process of treatments with drug and the probe, etc., which involve thorough washing away of the left overs with PBS or any buffer. Probe loading should be done before protocol for activating eNOS. It should be done in the presence of pluronics otherwise the probe will not accumulate in enough concentration intracellularly. Assay must be done in a buffer containing Ca2+ and glucose.
If fluorescence microscopic imaging is not an option, then flow cytometry is a fantastic way to go, yet not free from any hiccups.
If both of the above are not feasible, then I would stimulating NO generation and assay for cGMP and L-NAME-sensitive portion of this would represent NO-dependent cGMP generation.
If all of the above are not feasible, go for Griess and some journals are still accepting these studies.
Good luck.
Hi everyone,
I am trying to measure NO in endothelial cells, but have been impossible for me to see any change in fluorescence. I used 10 uM of DAF-FM DA prepared on a buffer solution without FBS and incubate for 30-60 min, then I washed the cells for 30 min. I applied bradykinin 10 uM but nothing happened.
Does anyone have any suggestions to improve the protocol?
Thanks in advance.
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