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AssayWise Letters Vol. 7(2)
Nitric Oxide (NO) is a free radical from the family of reactive nitrogen species (RNS). NO is an essential biological oxidant involved in a broad range of physiological and pathological processes. Dysregulation of NO production can cause damages to a wide array of biomolecules (e.g. proteins, enzymes, lipids and nucleic acids) and can cause various diseases ranging from stroke, heart disease, hypertension, neurodegeneration and erectile dysfunction, to gastrointestinal distress (Rose & Mascharak, 2008). Due to its extremely short lifetime and low steady-state concentration (Mayer, 2000), it remains challenging to selectively and quantitatively detect NO. The lack of quantitative measurements of intracellular NO has limited the understanding of the roles of NO in various biological processes.
To address this limitation, we have developed a series of NO fluorescence probes (Nitrixyte™) to detect NO production with high specificity and sensitivity. Compared to the widely used NO sensor, DAF-2 diacetate, Nitrixyte™ products significantly enhance the signal-to-background ratio in solution assays as well as in live cells. In this comparison study between Nitrixyte™ and DAF-2, two model systems of cell lines were investigated. In the first, NO was introduced exogenously by incubating live HeLa cells with the NO donor DEA NONOate. In the second system, NO was generated endogenously in live RAW 264.7 macrophage cells by drug treatment. As shown in Figure 1 and 2, the detection of exogenous NO in live cells was measured using a fluorescence microplate reader and visualized with a fluorescence microscope, respectively. The microplate reader data demonstrate that compared to DAF-2 diacetate, Nitrixyte™ Orange shows significantly lower background signal in control HeLa cells. More importantly, Nitrixyte™ Orange provides a 6-7 fold increase in signal-to-noise ratio as calculated by dividing the RFU of NONOate treated samples by the corresponding untreated control. The fluorescence images in Figure 2 and 3 also highlight the stronger staining and higher signal-to-noise ratio of Nitrixyte™ Orange over DAF-2 diacetate in detecting both exogenous NO and endogenous NO in live cells.
Figure 1 Microplate reader measurement of exogenous nitric oxide (NO) in HeLa cells upon DEA NONOate treatment (NO donor). Cells were incubated with AAT’s Nitrixyte™ Orange or DAF-2 diacetate at the same concentration for 30 minutes. The cells were then treated with or without 1 mM DEA NONOate at 37 ºC for 30 minutes. The fluorescence signals were measured with bottom read mode at Ex/Em=540/590 nm or Ex/Em=490/530 nm, respectively. The relative fluorescence signal intensity (A) and signal-to-noise ratio (B) were both measured.
Figure 2 Fluorescence images of exogenous nitric oxide (NO) detection in HeLa cells upon DEA NONOate treatment (NO donor). Cells were incubated with AAT’s Nitrixyte™ Orange (Left) or DAF-2 diacetate (Right) at the same concentration for 30 minutes. The fluorescence signals were measured using fluorescence microscope with a TRITC (Left) or FITC (Right) filter sets, respectively. All images were taken using the same exposure time.
Figure 3 Fluorescence images of endogenous nitric oxide (NO) detection in RAW 264.7 macrophage. Cells were incubated with AAT’s Nitrixyte™ Orange (Left) or DAF-2 diacetate (Right) at the same concentration, then treated with or without 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-arginine (L-Arg) at 37 ºC for 16 hours. The fluorescence signals were measured using fluorescence microscope with a TRITC (Left) or FITC (Right) filter set, respectively. All images were taken using the same exposure time.
Another unique advantage of Nitrixyte™ products is their capacity for multiplexing applications in flow cytometric analysis or fluorescence imaging. Through the combination of Nitrixyte™ products with reactive oxygen species (ROS) probes, researchers can simultaneously detect both NO and ROS in live cells. Figure 4 shows an example of applying both Nitrixyte™ Orange (Red) and Amplite™ ROS Green (Green) in macrophage cells. The cells were treated with 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-arginine (L-Arg) to stimulate endogenous NO. Cells were also treated with 50 µM pyocyanin (Pyo) at the same time to stimulate ROS generation. After 16 hour treatment, the fluorescence signals were measured using a fluorescence microscope. The red fluorescence from Nitrixyte™ Orange indicates the generation of endogenous NO in cells while the green fluorescence of Amplite™ ROS Green shows the increased level of total ROS after treatment. These results highlight the successful application of Nitrixyte™ Orange in multiplexing assay with ROS probes. The distinguished green and red fluorescence signal also indicate the high specificity of both Nitrixyte™ Orange and Amplite™ ROS Green.
Figure 4 Fluorescence images of simultaneous detection of intracellular nitric oxide (NO) and total ROS in RAW 264.7 macrophage. Cells were co-stained with Nitrixyte™ Orange (Red) and Amplite™ ROS Green (Green) after treatment with lipopolysaccharide (LPS), L-arginine (L-Arg) and pyocyanin (Pho) for 16 hours. The untreated control cells were stained under the same condition but without treatment. The fluorescence signals were measured using fluorescence microscope with TRITC (Nitrixyte™ Orange, Red) and FITC (Amplite™ ROS Green, Green) filter sets, respectively.
AAT Bioquest’s Nitrixyte™ fluorescence probes and related Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kits offer robust and sensitive methods to detect NO in live cells. Compared to the widely used DAF-2 diacetate, Nitrixyte™ products have a lower nonspecific staining in cells which contributes to a significantly higher signal-to-noise ratio. This distinct advantage allows Nitrixyte™ products to be combined with ROS probe for multiplex staining. It has been both challenging and important for researchers to investigate the co-existence and interaction of both RNS and ROS in live cells. Based on our data, Nitrixyte™ products offer the best analytical tools with highest sensitivity and specificity for the thorough understanding of NO in cellular redox regulation. They are applicable in fluorescence imaging, microplate reader and flow cytometer assays, allowing for simple and fast screening tests and will benefit both RNS and ROS chemical biology significantly.
Table 1. Product Ordering Information
|Product Name||Assay Target||Fluorescence||Cat. #||Instrument||Unit Size|
|Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit||Nitric Oxide||Orange||16350||Microplate Reader||200 Tests|
|16351||Flow Cytometer||100 Tests|
|Red||16356||Flow Cytometer||100 Tests|
|NIR||16359||Microplate Reader||200 Tests|
|16360||Flow Cytometer||100 Tests|
1.1 Simple Protocol for Imaging or Plate-reader Assays
- To stimulate endogenous NO, treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in cell culture medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of medium or compound buffer.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Nitrixyte™ working solution in the cell plate. Co-incubate cells with test compound and Nitrixyte™ working solution at 37 °C for desired period of time, protected from light.
Note 1:DO NOT remove the test compounds.
Note 2: Prepare Nitrixyte™ working solution according to the detailed protocol of each kit.
- To induce exogenous NO in living cells, please see examples in 1.3.
- Remove solution in each well. Add Assay Buffer II 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate.
- Monitor the fluorescence increase using microplate reader or take images using fluorescence microscope with the filter sets according to each kit.
1.2 Simple Protocol for Flow Cytometer Assays
- For each sample, prepare cells in 0.5 mL warm medium or buffer of your choice at a density of 5×105 -1×106 cells/mL.
- Add Nitrixyte™ probe into 0.5 mL cell suspension.
Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Nitrixyte™ probe.
- Incubate cells with test compounds and Nitrixyte™ probe at 37 ºC for a desired period of time to generate endogenous NO.
- To induce exogenous NO in living cells, please see examples in 1.3.
- Analyze cells with a flow cytometer with the channel setting according to each kit.
1.3 Examples of Generating Exogenous or Endogenous NO in Cells
- Exogenous NO model: Incubate cells with Nitrixyte™ working solution at 37 ºC for 30 minutes. Remove Nitrixyte™ working solution and incubate cells with 1 mM DEA/NONOate positive control working solution at 37 ºC for 30 minutes to generate exogenous nitric oxide.
- Endogenous NO model: Incubate Raw 264.7 cells with Nitrixyte™ working solution, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37 °C for 16 hours.
- Rose MJ and Mascharak PK. (2008) Fiat Lux: selective delivery of high flux of nitric oxide (NO) to biological targets using photoactive metal nitrosyls. Curr Opin Chem Biol 12(2), 238-244
- Mayer B. (2000) Nitic Oxide; Handbook of Experimental Pharmacology. Springer, Berlin.
- Vogel, S. N. (2000). LPS Another piece of the puzzle J. Endotoxm Res. 6, 295-300
- Hetrick EM, SchoenfischMH (2009) Analytical chemistry of nitric oxide. Annu RevAnal Chem 2:409–433.
- Nagano T, Yoshimura T (2002) Bioimaging of nitric oxide. Chem Rev 102:1235–1269
- Miller EW, Chang CJ (2007) Fluorescent probes for nitric oxide and hydrogen peroxide in cell signaling. Curr Opin Chem Biol 11(6):620–625.