Dax J2™ Sensors
Most cellular ROS are generated endogenously as byproducts of mitochondrial oxidative phosphorylation, or formed as intermediates of oxidoreductase enzymes and metal catalyzed oxidation. Since oxygen atoms contain two unpaired electrons in separate orbits of its outer electron shell, it is susceptible to radical formation. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including nitric oxide (NO).
The spectral properties of DAX-J2TM reagents. DAF-2 (Green), DAX-J2TM Orange (Orange), Red (Red) and IR (Dark Red) in PBS buffer (pH 7.2).
DAF-2 reagents are frequently used to detect nitric oxide (NO). However, DAF-2 diacetate is spontaneously hydrolyzed in cell culture media. The hydrolyzed DAF-2 is not cell-permeable, thus causing high assay background. DAX-J2™ probes are developed as excellent replacements for DAF-2 for the detection and bioimaging of NO. Compared to DAF-2 reagents, DAX-J2™ reagents have longer wavelengths and better stability.
The DAX-J2™ series universally demonstrate pH-independent fluorescent response and are highly specific to NO even in the presence of multiple reactive oxygen species (ROS).
- pH-independent spectral properties
- No esterase activity required for NO detection
- Multiple colors and the newly-added ratiometric option allow for compatibility with multiple dye-labeled antibodies for multicolor cell analysis
DAX-J2™ series are non-fluorescent reagents that can measure free NO and nitric oxide synthase (NOS) activity in living cells under physiological conditions. Once inside the cell the blocking groups on the DAX-J2 reagent are released to generate the highly fluorescent product upon NO oxidation. The majority of the dyes are cell-permeable, with the lone exception, DAX-J2™ IR, instead being water-soluble. The entire series exhibits pH-independent fluorescence.
Table 1. DAX-J2™ Nitric Oxide (NO) Sensors For DAX-J2™ Sensors
|16300||DAX-J2™ Orange||545||576||Yes||1 mg|
|16301||DAX-J2™ Red||588||610||Yes||1 mg|
|16302||DAX-J2™ IR||780||800||No||1 mg|
|16310||DAX-J2™ Ratio 580/460||420/540||460/580||Yes||1 mg|
- The excitation (Ex) and Emission (Em) wavelength in the above table are the chemical properties of the dyes. It is highly recommended to consult your instrument company with the proper Ex/Em wavelengths and filters to use. For example, when using a Gemini or FlexStation fluorescence microplate reader (Molecular Devices), one will need to use Ex/Em = 540/590 nm (cut off at 570 nm) for Cat #16300, and ratio of Ex/Em = 540/590 nm (cut off at 570 nm) vs. Ex/Em = 420/480 nm (cut off at 455 nm) for Cat #16310 respectively.
Fluorescence Spectrum Viewer
Need assistance selecting the best fluorophore for your experiment, use our Fluorescence Spectrum Viewer:
- View and compare fluorophores and fluorescent proteins for biological applications
- Check spectral compatibility
- Add multiple excitation and emission filters
- Save spectra configuration as a PNG or hyperlink
Other Series Kits Integrating DAX-J2™ Sensors
The Amplite™ and Cell Meter™ product series both include peroxynitrite kits utilizing the DAX-J2™ PON Green 99 sensor, which specifically reacts with peroxynitrite (ONOO-) with high selectivity over other reactive oxygen species (ROS) and reactive nitrogen species (RNS) to generate a bright green glow easily measured using the common FITC filter.
- For measurement of peroxynitrite in solution, the Amplite™ Fluorimetric Peroxynitrite Quantification Kit can be used either with a fluorescence microplate reader or spectrometer.
- For measurement of intracellular peroxynitrite, the Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (pictured right in Figure 2) works perfectly on multiple platforms including fluorescence imaging, flow cytometry and fluorescence microplate reader-based assays.
Fluorescence images of intracellular peroxynitrite in RAW 264.7 macrophage cells using Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16315). Raw 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. SIN-1 Treatment: Cells were co-incubated with DAX-J2™ PON Green and 100 µM SIN-1 at 37°C for 1 hour. Untreated control: The RAW 264.7 cells were incubated with DAX-J2™ PON Green without SIN-1 treatment. The fluorescence signals were measured using a fluorescence microscope with a FITC filter