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Nitric Oxide Detection
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Hydrogen Peroxide Detection
Catalase Detection
Peroxidase Detection
Hydroxyl Radical Detection
Superoxide Detection
SOD Detection
Xanthine Assay
Hypochlorite Assay
Total ROS Detection
Thiol Detection
Nitric Oxide Detection
AssayWise Letters 2015, Vol. 4(2)
Nitric oxide (NO) free radical is an important cellular signaling molecule involved in many physiological and pathological processes. It is an important biological regulator and is therefore a fundamental component in the fields of neuroscience, physiology, and immunology. Nitric oxide is a powerful vasodilator with a short half-life of a few seconds in the blood. Long-known pharmaceuticals, such as nitroglycerine and amyl nitrite, were discovered, more than a century after their first use in medicine, to be active through the mechanism of being precursors to nitric oxide. Low levels of nitric oxide production are important in protecting organs, such as the liver, from ischemic damage.
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 (Cat# 16301) 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 most flow cytometers and fluorescence microscopes equipped with the filter set of Texas Red®.
DAX-J2™ Orange (Cat# 16300) 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®/TRITC.
DAX-J2™ IR (Cat# 16302) is a new fluorogenic NO sensor that has near infrared fluorescence. DAX-J2™ IR reagent is highly water-soluble. It enables NO detection in vivo using IVIS® Imaging System(PerkinElmer) or Kodak Image Station.
DAX-J2™ Ratio 580/460 (Cat# 16310) is a new nitric oxide (NO) sensor recently developed by AAT Bioquest. It is a cell permeable reagent that can measure free NO and nitric oxide synthase (NOS) activity in living cells in a ratiometric mode. Once inside the cell, the blocking groups on the DAX-J2™ reagent are released to induce fluorescence ratio changes at wavelengths of 580 nm and 460 nm upon NO oxidation. The fluorescence intensities at 580 nm and 460 nm can be detected using the filter sets of Cy3®/TRITC and BD Horizon™ V450/Pacific Blue. Most of flow cytometers and fluorescence microscopes are equipped with these two filter sets.
DAX-J2™ Ratio 580/460 has distinct advantages for NO detection over the popular DAF-2 NO probe: 1). DAX-J2™ Ratio 580/460 does not require esterase activity for NO detection. DAF-2 requires intracellular esterases to cleave its acetate groups for detecting NO activity. 2). DAX-J2™ product exhibits pH-independent fluorescence while DAF-2 has its fluorescence highly affected by pH. 3). DAX-J2™ Ratio 580/460 can be monitored in a ratiometric mode.
Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO is rapidly oxidized and exists in relatively low concentration. It has been challenging to detect and understand the role of NO in biological systems. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits provide a robust tool to monitor intracellular NO level in live cells.
Nitrixyte™ Orange and Nitrixyte™ Red are developed as excellent replacements for DAF-2 for the detection and imaging of free NO in cells. Compared to the widely used DAF-2 probes, Nitrixyte™ Orange and Nitrixyte™ Red have better photostability and enhanced cell permeability. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits (Cat# 16350 & 16351) use Nitrixyte™ Orange that reacts with NO to generate a bright orange fluorescent product. The NO-generated product of Nitrixyte™ Orange has spectral properties similar to those of Cy3® and TRITC. Nitrixyte™ Orange can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy3® or TRITC. Kit 16350 is optimized for fluorescence imaging and microplate reader applications. Kit 16351 is optimized for flow cytometry applications.
Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit 16356 uses Nitrixyte™ Red that reacts with NO to generate a bright red fluorescent product. The NO-generated fluorescent product of Nitrixyte™ Red has spectral properties similar to those of Texas Red®. Nitrixyte™ Red can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Texas Red®. Kit 16356 is optimized for flow cytometry applications.
Key Features of DAX-J2™ NO Detection Probes:
- 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.
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.
The spectral properties of DAX-J2™ reagents. DAF-2 (Green), DAX-J2™ Orange (Orange, Cat# 16300), Red (Red, Cat# 16301) and IR (Dark Red, Cat# 16302) in PBS buffer (pH 7.2).
DAX-J2™ Red (Cat# 16301) 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 most flow cytometers and fluorescence microscopes equipped with the filter set of Texas Red®.
DAX-J2™ Orange (Cat# 16300) 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®/TRITC.
Fluorescence responses of DAX-J2™ Orange (5 µM, Cat# 16300) to different reactive oxygen species (1 mM) in PBS buffer (pH 7.2). The fluorescence intensities were measured at Ex/Em = 540/570 nm.
DAX-J2™ IR (Cat# 16302) is a new fluorogenic NO sensor that has near infrared fluorescence. DAX-J2™ IR reagent is highly water-soluble. It enables NO detection in vivo using IVIS® Imaging System(PerkinElmer) or Kodak Image Station.
DAX-J2™ Ratio 580/460 (Cat# 16310) is a new nitric oxide (NO) sensor recently developed by AAT Bioquest. It is a cell permeable reagent that can measure free NO and nitric oxide synthase (NOS) activity in living cells in a ratiometric mode. Once inside the cell, the blocking groups on the DAX-J2™ reagent are released to induce fluorescence ratio changes at wavelengths of 580 nm and 460 nm upon NO oxidation. The fluorescence intensities at 580 nm and 460 nm can be detected using the filter sets of Cy3®/TRITC and BD Horizon™ V450/Pacific Blue. Most of flow cytometers and fluorescence microscopes are equipped with these two filter sets.
Fluorescence responses of DAX-J2™ Ratio 580/460 (2 µM, Cat# 16310) to different reactive oxygen species (1 mM) in PBS buffer (pH = 7.2). The fluorescence intensities were measured at 580 nm and 460 nm respectively.
DAX-J2™ Ratio 580/460 has distinct advantages for NO detection over the popular DAF-2 NO probe: 1). DAX-J2™ Ratio 580/460 does not require esterase activity for NO detection. DAF-2 requires intracellular esterases to cleave its acetate groups for detecting NO activity. 2). DAX-J2™ product exhibits pH-independent fluorescence while DAF-2 has its fluorescence highly affected by pH. 3). DAX-J2™ Ratio 580/460 can be monitored in a ratiometric mode.
Table 1. DAX-J2™ Nitric Oxide (NO) Sensors For DAX-J2™ Sensors
| Cat# ▲ ▼ | Product Name ▲ ▼ | Ex ▲ ▼ | Em ▲ ▼ | Cell Permeability ▲ ▼ | Unit Size ▲ ▼ |
| 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 |
Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO is rapidly oxidized and exists in relatively low concentration. It has been challenging to detect and understand the role of NO in biological systems. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits provide a robust tool to monitor intracellular NO level in live cells.
Detection of exogenous nitric oxide (NO) in cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat#16350). CHO-K1 and HeLa cells were seeded at 50,000 cells/well/100 µL overnight in a 96-well black wall/clear bottom plate. Cells were incubated with Nitrixyte™ Orange working solution at 37 °C for 30 minutes. The cells were treated with or without 1mM DEA NONOate at 37 °C for 30 minutes. The fluorescence signals were monitored at Ex/Em = 540/590 nm (cut off at 570 nm) with bottom read mode.
Nitrixyte™ Orange and Nitrixyte™ Red are developed as excellent replacements for DAF-2 for the detection and imaging of free NO in cells. Compared to the widely used DAF-2 probes, Nitrixyte™ Orange and Nitrixyte™ Red have better photostability and enhanced cell permeability. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits (Cat# 16350 & 16351) use Nitrixyte™ Orange that reacts with NO to generate a bright orange fluorescent product. The NO-generated product of Nitrixyte™ Orange has spectral properties similar to those of Cy3® and TRITC. Nitrixyte™ Orange can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy3® or TRITC. Kit 16350 is optimized for fluorescence imaging and microplate reader applications. Kit 16351 is optimized for flow cytometry applications.
Fluorescence images of endogenous nitric oxide (NO) measurement in RAW 264.7 macrophage cells using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat# 16350). Raw 264.7 cells were seeded at 100,000 cells/well/100 µL overnight in a 96-well black wall/clear bottom plate. Cells were incubated with Nitrixyte™ Orange, and treated with (Right) or without (Left) 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) at 37 °C for 16 hours. The fluorescence signals were measured using fluorescence microscope with a TRITC filter.
Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit 16356 uses Nitrixyte™ Red that reacts with NO to generate a bright red fluorescent product. The NO-generated fluorescent product of Nitrixyte™ Red has spectral properties similar to those of Texas Red®. Nitrixyte™ Red can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Texas Red®. Kit 16356 is optimized for flow cytometry applications.
Left: Detection of exogenous nitric oxide (NO) in Jurkat cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat# 16351). Cells were incubated with Nitrixyte™ Orange at 37 °C for 30 minutes and washed twice with assay buffer. The cells were treated with (Red) or without (Blue) 1mM DEA NONOate at 37 °C for 30 minutes. The fluorescence signals were monitored in FL2 channel. Right: Detection of exogenous nitric oxide (NO) in Jurkat cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Activity Assay Kit (Cat# 16356). Cells were incubated with Nitrixyte™ Red at 37 °C for 30 minutes. The cells were treated with (Red) or without (Blue) 1mM DEA NONOate at 37 °C for 2 hours. The fluorescence signals were monitored using a flow cytometer (BD FACSCalibur™) in FL4 channel.
Table 2. Intracellular Nitric Oxide (NO) Assay Kits
| Cat No. ▲ ▼ | Product Name ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ |
| 16351 | Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Flow Cytometry* | 488 | 590 | 100 Tests |
| 16350 | Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Microplate Reader* | 540 | 590 | 200 Tests |
| 16356 | Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Red Fluorescence Optimized for Flow Cytometry* | 630 | 660 | 100 Tests |