Amplite® Colorimetric Nitrite Quantification Assay Kit

Product key features

The Amplite® Colorimetric Nitrite Quantification Assay Kit provides a simple and sensitive solution for measuring nitrite levels in biological samples.

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® Colorimetric Nitrite Quantification Assay Kit offers a sensitive colorimetric method for quantifying nitrite levels in various biological samples. Nitrite serves as a key indicator of nitric oxide (NO) production through the nitrate-nitrite-NO pathway, which plays a pivotal role in vasodilation, immune function, and cellular signaling. Precise nitrite quantification is crucial for studies investigating nitric oxide biology and its role in both physiological and pathological processes.

This kit employs a colorimetric detection strategy where nitrite reacts with a developer to produce a visible color change. The resulting color intensity, measurable using a standard absorbance microplate reader, is directly proportional to the nitrite concentration in the sample. The assay’s reagents are optimized for straightforward preparation, enabling rapid, reliable results with minimal sample processing. The kit is compatible with various sample types, such as cell culture media and serum, making it an essential tool for researchers examining nitric oxide metabolism and related biological mechanisms.

Example protocol

AT A GLANCE

For 96 well plates:

Prepare test samples along with serially diluted nitrite standards (75 μL).
Add Griess Reagent working solution (75 μL).
Incubate at RT for 10 minutes.
Measure the absorbance at 548 nm.

For spectrophotometry cuvettes:

In a 1 cm path length cuvette, mix test samples or serially diluted nitrite standards with Griess Reagents:
- 2.6 mL of deionized water
- 100 µL of Griess Reagent
- 300 µL of the nitrite-containing sample
Incubate at RT for 10 minutes.
Measure the absorbance at 548 nm.

Important note: Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/21666

Nitrite Standard Dilution (For absorbance plate reader)

Add 2 μL of 50 mM nitrite standard solution (component B) into 1 mL of distilled water to get 100 µM nitrite standard solution (STD7). Take 500 μL (STD7) and perform 2X serial dilutions in distilled water to get serially diluted nitrite from 50 µM, 25 µM,12.5 µM, 6.25 µM, 3.13 µM and 1.56 µM (STD6 to STD1).

PREPARATION OF WORKING SOLUTION

Griess Reagent Working Solution

Add 1 mL Griess Reagents (Component A) to 6.5 mL of distilled water. Mix well, protected from light.

Note: Working solution should be prepared fresh for each experiment.

SAMPLE EXPERIMENTAL PROTOCOL

For Absorbance Microplate Reader

Prepare Nitrite Standards (STD1-7), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 37.5 µL of reagent per well instead of 75 µL.
Add 75 µL of Griess Reagent working solution to each well of blank control, nitrite positive control and test samples. For a 384-well plate, add 37.5 µL of nitrite working solution into each well instead.
Incubate at RT for 10 minutes, protected from light.
Measure absorbance at 548 nm.

Table 1. Layout of Nitrite standards and test samples in a clear bottom 96-well microplate. STD= Nitrite Standards (STD1-STD7, 1.55 to 100 µM), BL=Blank Control, TS=Test Samples.

Cell content	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

Note: Nitrite concentrations in the test samples (TS) should be within the linear range of the assay (~1-100 µM).

Table 2. Reagent composition for each well.

Well	Volume	Reagent
STD1-STD7	75 µL	Serial Dilutions (1.55 to 100 µM)
BL	75 µL	Distilled water
TS	75 µL	Test Sample

For Spectrophotometry Assay:

Nitrite Standard Dilution:

Add 4 μL of 50 mM nitrite standard solution (component B) into 2mL of distilled water to get 100 µM nitrite standard solution (STD7). Take 1 ml (STD7) and perform 2X serial dilutions in distilled water to get serially diluted Nitrite from 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.13 µM and 1.56 µM (STD6 to STD1).

Assay protocol:

Add the following in a cuvette with 1 cm path length:
- 2.6 mL of deionized water
- 100 µL of Griess Reagent (Component A)
- 300 µL of the nitrite-containing sample
Note: Nitrite concentrations in the test samples (TS) should be within the linear range of the assay (1-100 µM).
Incubate RT for 10 min.
Measure absorbance at 548 nm.

References

View all 50 references: Citation Explorer

Rapid and dual-mode nitrite detection with improved sensitivity by nanointerface-regulated ultrafast Griess assay.
Authors: Sun, Jingbo and Long, Tiantian and Chen, Zihan and Luo, Hongmei and Cao, Jiafeng and Xu, Dong and Yuan, Zhiqin
Journal: Analytica chimica acta (2025): 343524

Rapid Griess assay (RGA): a chairside test for ex vivo semi-quantitative oral nitrite measurement and in vitro assessment of nitrite production by oral bacteria.
Authors: Mavropoulos, Simeon K B and Zaiton, Rabi and Basic, Amina and Dahlén, Gunnar
Journal: Journal of oral microbiology (2025): 2517039

Presumptive identification of nitrite by Griess reagent test strips-Case reports of fatal poisoning with sodium nitrite.
Authors: Zhang, Man and Truver, Michael T and Hoyer, Jennifer L and Chronister, Chris W and Goldberger, Bruce A
Journal: Journal of analytical toxicology (2023): 746-749

Griess-doped polyvinyl alcohol thin film for on-site simultaneous sample preparation and nitrite determination of processed meat products.
Authors: Taweekarn, Tarawee and Wongniramaikul, Worawit and Boonkanon, Chanita and Phatthanawiwat, Kharittha and Pasitsuparoad, Pakorn and Ritchie, Raymond J and Choodum, Aree
Journal: Food chemistry (2022): 133085

Improving Sustainability of the Griess Reaction by Reagent Stabilization on PDMS Membranes and ZnNPs as Reductor of Nitrates: Application to Different Water Samples.
Authors: Hakobyan, Lusine and Monforte-Gómez, Belén and Moliner-Martínez, Yolanda and Molins-Legua, Carmen and Campíns-Falcó, Pilar
Journal: Polymers (2022)

Page updated on July 20, 2025

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