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Detection and Bioimaging of Nitric Oxide (NO) Using Multicolor DAX " J2™ Reagents


The spectral properties of DAX-J2™ reagents. DAF-2 (Green), DAX-J2™ 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. AAT Bioquest offers three distinct DAX-J2™ multicolor imaging reagents for NO detection.

DAX-J2™ Red is a new nitric oxide (NO) sensor recently developed by AAT Bioquest. It is a non-fluorescent cell permeable reagent 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 a highly red fluorescent product upon NO oxidation. The DAX-J2™ fluorescent product can be detected using the filter set of Texas Red® that is equipped with most of flow cytometers and fluorescence microscopes.

DAX-J2™ Orange generates a bright orange fluorescent product that has spectra properties similar to those of Cy3® and TRITC. DAX-J2™ Orange can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy3® and TRITC.

DAX-J2™ IR is a new fluorogenic NO sensor that has near infrared fluorescence. This DAX-J2™ IR reagent is highly water-soluble. It enables NO detection in vivo using IVIS® Imaging System(Caliper) or Kodak Image Station.


Fluorescence response of DAX-2J™ Orange (5 µM) to different reactive oxygen species (1 mM) in PBS buffer (pH 7.2). The fluorescence intensities were measured with Ex/Em = 540/570 nm.

Key Features of DAX-J2™ NO Detection Reagents:

  • No esterase activity required for NO detection
  • pH-independent spectral properties
  • Much more photostable than DAF-2
  • More tolerant to cell medium hydrolysis than DAF-2
  • Compatible with GFP cell lines or the applications that use FITC-labeled antibodies for multicolor cell analysis

 

Table 1. DAX-J2™ Nitric Oxide (NO) Sensors For DAX-J2™ Sensors

Cat#
Product Name
Ex
Em
Cell Permeability
Unit Size
Price
16300DAX-J2™ Orange545576Yes1 mg$162
16301DAX-J2™ Red588610Yes1 mg$162
16302DAX-J2™ IR780800No1 mg$327
16310DAX-J2™ Ratio 580/460420/540460/580Yes1 mg$437