I want to detect nitric oxide (NO) with flow cytometry. I found two agent which are 4,5-Diaminofluorescein diacetate solution and 5,6-Diaminofluorescein diacetate- Sigma-Aldrich. Which is the best? I will work with MDSC and detect their ıntracelluler NO production. I am waiting your suggestion.
http://www.sigmaaldrich.com/catalog/product/sigma/50277?lang=en®ion=TR
http://www.sigmaaldrich.com/catalog/product/sigma/d225?lang=en®ion=TR
I recommend DAF-FM diacetate, in place of the two from Sigma.
The DAF probes were originally developed by Tetsuo Nagano's group at Tokyo University. The detection mechanism is the same for all the DAF probes. DAF-FM however, is protected as a difluoro probe (against nonspecific oxidation). The diacetate is for improving probe retention in cells.
Note that after incubation (typically 15-20 min), the probe requires a wash step and further incubation in buffer for the fluorescent product to mature.
Thermo/Invitrogen sells the probe in small aliquots. Each vial is enough for an experiment.
https://www.thermofisher.com/order/catalog/product/D23842
Good luck.
Yes, in agreement with Nai-Kei, flow cytometry would give more reliable results than plate reader or fluorescence microscopy. I have used microscopy for a longer time and currently using flow as we are dealing with nonadherent cells. I am not sure about the cell type you are using, and I did have to change the protocol, incubation period and concentration, to fit with our specific experimental protocol.
DAF-FM diacetate is the one we have been using, again the cell type determines how long is the incubation, you need to play with it for a couple of times.
Good luck...
We have demonstrated that intracellular nitric oxide (NO) can be detected and measured directly in human spermatozoa by flow cytometry using the fluorescent probe 4,5-diaminofluorescein-2/diacetate (10 microM).
Int J Androl. 2006 Oct;29(5):564-7
DAF-FM diacetate, from Invitrogen, has worked beautifully in our hands in primary cells, including microglia.
I WOULD LIKE TO MEASURE NITRIC OXIDE PRODUCED BY SEEDS USING FLOW CYTOMETRY.wILL YOU ,PLEASE, BE SO KIND TO GIVE ME SOME ADVICE?
THANK YOU IN ADVANCE
I was following the informative discussion. Please provide me the protocol/reference article for NO measurement using DAF-FM using Tecan plate reader. Thank you
I think the best probe to be used is DAF-FM DA.
The optimized protocol for intracellular NO analysis involves DAF-FM DA at 10 μM for 60 min to determine the mean fluorescence intensity. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in cells, while the NO scavenger compound significantly will decrease the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.
Gourav, how soon can you observe the fluorescence change upon adding donor?
I am using DAF-FM from Thermofisher to observe the fluorescence difference of endothelial cells between flow and non-flow conditions. I can always see the fluorescence signal even with .5 uM DAF-FM, but the problem is that I can't see any difference between two. Do you guys have any suggestions?
- Badges
- Science method