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Amplite® Fluorimetric Nitrite Quantification Assay Kit *Blue Fluorescence*
Product key features
- Sensitive Detection: Precise and reliable nitrite quantification.
- Optimized Reagents: Includes all necessary components for efficient and reproducible results.
- Simple Protocol: Quick and straightforward procedure with minimal hands-on time.
- High Compatibility: Suitable for a variety of biological samples, including cell culture supernatants and serum.
Product description
The Amplite® Fluorimetric Nitrite Quantification Assay Kit is a highly sensitive and reliable method for measuring nitrite levels in biological samples, including cell culture supernatants and serum. Nitrite is a crucial precursor in the nitrate-nitrite-nitric oxide (NO) pathway, which plays a significant role in vasodilation, immune responses, and cellular signaling. Accurate quantification of nitrite is essential for researchers studying NO metabolism and its implications in various physiological and pathological processes.
This assay employs a fluorescence-based detection method, wherein nitrite reacts with a developer reagent to produce a fluorescent signal. The fluorescence intensity is directly proportional to the nitrite concentration, allowing for precise quantification using a fluorescence microplate reader. The kit includes optimized reagents for efficient and reproducible results, featuring a simple protocol with minimal hands-on time. Its high compatibility with various biological samples makes it an excellent tool for researchers investigating nitric oxide production and its role in health and disease.
Example protocol
AT A GLANCE
- Prepare test samples along with serially diluted nitrite standards (50 μL).
- Add Nitrite Blue working solution (50 μL).
- Incubate at RT for 10 minutes.
- Add 15 μL of Nitrite Developer.
- Measure the Fluorescence at Ex/Em=365/450 nm, Cut-off=435 nm.
Thaw all the kit components at room temperature before starting the experiment and centrifuge the vials briefly before opening.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/21667
PREPARATION OF WORKING SOLUTION
Add 20 µL of Nitrite Blue (Component A) to 5 mL of Nitrite Assay Buffer (Component B). Mix well, protected from light.
Note: This working solution is enough for one 96-well plate. It is unstable at room temperature, and should be used promptly within 2 hours and avoid exposure to light. Alternatively, one can prepare the needed working solution proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1: Layout of Nitrite standards and test samples in a clear bottom 96-well microplate. STD = Nitrite Standards (STD 1-STD 7, 1.55 to 100µM), BL= Blank Control, TS = Test Samples.
BL | BL | Positive Control | TS |
STD 1 | STD 1 | ... | ... |
STD 2 | STD 2 | ... | ... |
STD 3 | STD 3 | ... | |
STD 4 | STD 4 | ||
STD 5 | STD 5 | ||
STD 6 | STD 6 | ||
STD 7 | STD 7 |
Table 2: Reagent composition for each well.
| Well | Volume | Reagent |
| STD1-STD7 | 50 µL | Serial Dilutions (1.55 to 100 µM) |
| BL | 50 µL | dd H2O |
| TS | 50 µL | Test Sample |
- Prepare nitrite standards (STD 1-7), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 uL of reagent per well instead of 50 uL.
- Add 50 uL of Nitrite Blue Working Solution to each well of blank control, standard and test samples. For a 384-well plate, add 25 uL of nitrite Working Solution into each well instead.
- Incubate at RT for 10 minutes, protected from light.
- Add 15 µL Nitrite Developer (Component C) to each well of blank control, standard and test samples. For a 384-well plate, add 7.5 uL of Nitrite Developer (Component C) into each well instead.
- Incubate at RT for 5 min.
- Measure fluorescence intensity with a fluorescence microplate reader at Ex/Em = 365/450 nm, Cut-off = 435 nm.
References
Authors: Giroux, Lauriane and Isayenka, Iauhenia and Lerat, Sylvain and Beaudoin, Nathalie and Beaulieu, Carole
Journal: Frontiers in microbiology (2024): 1466927
Authors: Do, Thien D and Pifer, Ashley D and Wahman, David G and Hickman, Rylie N and Chimka, Justin R and Fairey, Julian L
Journal: Water research (2023): 119430
Authors: Zhang, Kai and Wang, Ming-Xin and Zeng, Hong-Yan and Li, Zhen
Journal: Mikrochimica acta (2022): 411
Authors: Silva-Silva, João Victor and Moragas-Tellis, Carla Junqueira and Chagas, Maria do Socorro Dos Santos and de Souza, Paulo Victor Ramos and de Souza, Celeste da Silva Freitas and Hardoim, Daiana de Jesus and Taniwaki, Noemi Nosomi and Moreira, Davyson de Lima and Dutra Behrens, Maria and Calabrese, Kátia da Silva and Almeida-Souza, Fernando
Journal: Frontiers in pharmacology (2021): 703985
Authors: Zakaria, Nur Nadiah and Malahubban, Masnindah and Fakurazi, Sharida and And, Wong Sie Chuong and Rajaee, Amy Halimah
Journal: Tropical life sciences research (2021): 145-162
Safety data sheet