DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate) (Invitrogen™)

DAF-FM is a reagent that is used to detect and quantify low concentrations of nitric oxide (NO). It is essentially nonfluorescent until it reacts with NO to form a fluorescent benzotriazole. DAF-FM fluorescence can be detected by any instrument that can detect fluorescein, including flow cytometers, microscopes, fluorescent microplate readers and fluorometers.

DAF-FM Specifications:
• Ex/Em of DAF-FM: ∼495/515 nm
• Lyophilized product should be dissolved using DMSO and then added to an aqueous buffer to create a working solution
• DAF-FM diacetate is cell permeant and passively diffuses across cellular membranes; once inside the cell, it is converted to a cell-impermeant form
• Buffers containing bovine serum albumin (BSA) and phenol red may affect the fluorescence and should be used with caution
• The fluorescence quantum yield of DAF-FM is ∼0.005, but increases about 160-fold, to ∼0.81, after reacting with NO

Applications of DAF-FM:
• Assessment of NO production in transaldolase-deficient lymphoblasts by flow cytometry
• Detection of NO accumulation in embryonic cortical neurons following neurotrophin stimulation
• in vivo imaging of NO in zebrafish
• Intravital microscopic detection of NO generation associated with angiogenesis in mice
• Quantitation of ATP-induced NO release in rabbit platelets

Important Advantages of DAF-FM Over DAF-2
The spectra of the NO adduct of DAF-FM are independent of pH above pH 5.5. Also, the NO adduct of DAF-FM is significantly more photostable than that of DAF-2, which means additional time for image capture. Finally, DAF-FM is a more sensitive reagent for NO than is DAF-2 (NO detection limit for DAF-FM ∼3 nM versus ∼5 nM for DAF-2).

Probes for Nitric Oxide Research
We offer several products for the detection of reactive oxygen species (ROS) and nitric oxide (NO). Review these products in Probes for Nitric Oxide Research—Section 18.3 in the Molecular Probes™ Handbook.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.