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Cat. No.: NOAK-100 (for >100T)

Cat. No.: NOAK-500 (for >500T)

Description

Nitric Oxide Assay Kit is a reagent kit that uses DAF-FM DA as a fluorescent probe to quickly and sensitively detect changes in nitric oxide levels within cultured cells. This kit can be used with various fluorescence detection equipment, including fluorescence microscopes, laser confocal microscopes, flow cytometers, and fluorescence microplate readers.

Nitric oxide (NO) is an inorganic nitrogen oxide compound. As a reactive free radical, it can rapidly diffuse through biological membranes due to its small molecular weight and lipophilicity. NO acts as a signaling molecule, transmitting signals produced by one cell to surrounding cells. It has functions such as vasodilation, immune regulation, inhibition of inflammatory responses, participation in the regulation of neuronal excitability and neurotransmitter release, DNA repair and protection, and plays an indispensable role in important physiological processes in the nervous system, immune system, cardiovascular system, and cell apoptosis1.

DAF-FM DA, also known as 3-Amino, 4-aminomethyl 2’,7’-difluorescein, diacetate, or DAF-FM diacetate or 4-Amino, 5-aminomethyl-2’,7’-difluorescein, diacetate, is a new generation fluorescent probe for the quantitative detection of nitric oxide. Compared to the previously commonly used nitric oxide fluorescent probe DAF-2 diacetate, DAF-FM DA has several improvements. Firstly, the fluorescent product formed by the reaction with nitric oxide is less affected by pH compared to DAF-2 diacetate, and is not affected by pH values greater than 5.5. Secondly, the fluorescence produced by DAF-FM DA is more stable and less prone to quenching, making it easier to detect. Additionally, DAF-FM DA has a higher sensitivity for nitric oxide detection, with detection sensitivity nearly twice that of DAF-2 diacetate under the same conditions, and a minimum detection concentration of 3 nM.

Components

• DAF-FM DA (5mM)
• Detection Buffer
• NOup (200X)

The kit provides DAF-FM DA as a 5 mM stock solution. The final concentration of DAF-FM DA is typically 1-10 μM, with the optimal recommended final concentration being 5 μM.

The kit also includes a positive control reagent, NOup, which induces nitric oxide production in cells. NOup is provided as a 200X compound mixture, with a recommended final concentration of 0.5-2X, and the optimal recommended final concentration being 1X. Additionally, the kit includes a detection buffer for more convenient use.

This kit is suitable for detecting intracellular nitric oxide levels and can be used for real-time detection. If cells are collected and then loaded with the probe, the small package can typically detect at least 100 samples, and the medium package can detect at least 500 samples. When the detection system volume per well in a 6-well plate is 1 ml, the small and medium packages can detect approximately 100 and 500 times, respectively.

Principle

DAF-FM DA can permeate cell membranes. Once inside the cell, it is catalyzed by intracellular esterases to form DAF-FM, which cannot cross the cell membrane. DAF-FM itself has very weak fluorescence, but it produces strong fluorescence upon reacting with nitric oxide, with an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Any instrument capable of detecting fluorescein green fluorescence, including fluorescence microscopes, laser confocal microscopes, flow cytometers, fluorescence spectrophotometers, or fluorescence microplate readers, can be used to detect this fluorescent probe.

Storage Conditions

Store at -20ºC, valid for one year. DAF-FM DA (5 mM) should be protected from light.

Precautions

• For the first use, aliquot DAF-FM DA (5 mM) and NOup (200X) and store at -20ºC to avoid repeated freeze-thaw cycles.
• Fluorescent probes should be prepared fresh before use. The prepared DAF-FM DA staining working solution should be used immediately and cannot be stored for later use.
• DAF-FM DA (5 mM) may solidify and stick to the bottom, walls, or cap of the centrifuge tube at 4ºC, in an ice bath, or at other low temperatures. It can be used after warming in a 20-25ºC water bath until fully dissolved.
• BSA and phenol red interfere with the detection of DAF-FM DA and should be avoided.
• Fluorescent dyes are prone to quenching; therefore, protect from light as much as possible to slow down fluorescence quenching.
• This kit is only suitable for detecting live cells or tissues and cannot be used for fixed or frozen cell or tissue samples.
• The detection buffer is filtered and sterilized. Avoid microbial contamination during use, as it may affect staining results. If the detection buffer becomes cloudy or shows signs of microbial contamination, it should not be used.
• When using a fluorescence microplate reader, use black or white plates suitable for fluorescence detection.
• This product is for scientific research use only by professionals. It is not intended for clinical diagnosis or treatment, nor for use in food or drugs, and should not be stored in ordinary residences.
• For your safety and health, please wear a lab coat and disposable gloves when handling.