Jan 15, 2026
Nitrite test with the Griess reagent system
- Mario Alberto Bautista-Carro1
- 1Cinvestav
External link: https://doi.org/10.1371/journal.pone.0339028
Protocol Citation: Mario Alberto Bautista-Carro 2026. Nitrite test with the Griess reagent system. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31ypel25/v1
Manuscript citation:
Bautista-Carro MA, Sánchez-Teoyotl P, Juárez-Serrano D, Iannitti T, Díaz A, Flores G, Morales-Medina JC (2026) Olfactory bulbectomy induces neurobiological alterations in the prefrontal cortex and hyperlocomotion in male rats. PLOS One 21(1). doi: 10.1371/journal.pone.0339028
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2025
Last Modified: January 15, 2026
Protocol Integer ID: 229344
Keywords: Determination of nitrites, Spectrophotometry, Griess reaction, Nitric oxide, nitrite test with the griess reagent system, nitrite test, evaluating nitrite level, nitrite level, griess reagent, griess reagent system, nitrite, griess method, napthylethylenediamine dihydrochloride
Abstract
This protocol describes the procedures for evaluating nitrite levels using the Griess method. The Griess reagent is composed of sulfanilamide, N-1-napthylethylenediamine dihydrochloride (NED) under acidic conditions (phosphoric acid).
Guidelines
We use a Griess reagent system.
Materials
Rodent Guillotine
Phosphate buffer solution
Laboratory centrifuge
Micropipette
96-well plate
Spectrophotometer
Griess Reagent System from Promega
Troubleshooting
Before start
Verify that the required materials and equipment are available.
Tissue preparation
Decapitate rats and immediately remove the brains.
Dissect the brain region of interest and homogenize in 3 ml of ice-cold 0.1 M phosphate buffer solution (PBS), pH 7.4.
Centrifuge the homogenate at 12,500 rpm (4 °C).
Collect the supernatant and store it at -70 °C.
Prepare reference curve in the same matrix or buffer used for experimental samples.
Prepare 1ml of a 100μM nitrite working solution by diluting the supplied 0.1M nitrite standard 1:1,000 in the same buffer used for the test samples.
Assign 3 adjacent columns (24 wells) of a 96-well plate to generate the nitrite standard reference curve. Add 50μl of the buffer into the wells in rows B–H.
Dispense 100μl of the 100μM nitrite solution into the 3 wells in row A.
Carry out 6 serial twofold dilutions (50μl per well) in triplicate down the plate to produce the nitrite standard reference curve (100, 50, 25, 12.5, 6.25, 3.13 and 1.56μM). Discard 50μl from the final dilution (1.56μM). Leave the last set of wells without nitrite (0μM).
Note: Each well should contain a final volume of 50μl, covering a concentration range of 0–100μM.
Nitrite Measurement (Griess Reaction)
Add 50 μl of each experimental sample into wells, in triplicate.
Add 50 μl of Sulfanilamide Solution to all wells (samples and nitrite standard curve).
Incubate for 10 min at room temperature under diminished light.
Add 50 μl of NED Solution to each well.
Incubate for 10 min at room temperature under diminished light. A purple to magenta coloration will appear rapidly.
Measure absorbance at 540 nm using a spectrophotometer.
Nitrite Concentration Determination in Experimental Samples
Plot the average absorbance value of each Nitrite Standard concentration as a function of "Y" with nitrite concentration as a function of "X" to create a Nitrite Standard reference curve.
Calculate each experimental sample's average absorbance value. By comparing it to the Nitrite Standard reference curve, ascertain its concentration. The results are expressed in μM of NO2 per mg of protein.
Protocol references
Díaz, A., Treviño, S., Guevara, J., Muñoz-Arenas, G., Brambila, E., Espinosa, B., Moreno-Rodríguez, A., Lopez-Lopez, G., Peña-Rosas, U., Venegas, B., Handal-Silva, A., Morán-Perales, J. L., Flores, G., & Aguilar-Alonso, P. (2016). Energy Drink Administration in Combination with Alcohol Causes an Inflammatory Response and Oxidative Stress in the Hippocampus and Temporal Cortex of Rats. Oxidative medicine and cellular longevity, 2016, 8725354. https://doi.org/10.1155/2016/8725354
González-Cano, S. I., Camacho-Abrego, I., Diaz, A., de la Cruz, F., Morales-Medina, J. C., & Flores, G. (2021). Prenatal exposure to propionic acid induces altered locomotion and reactive astrogliosis in male rats. Journal of chemical neuroanatomy, 117, 102011. https://doi.org/10.1016/j.jchemneu.2021.102011
Lima-Castañeda, L. Á., Bringas, M. E., Aguilar-Hernandez, L., Garcés-Ramírez, L., Morales-Medina, J. C., & Flores, G. (2023). The antipsychotic olanzapine reduces memory deficits and neuronal abnormalities in a male rat model of Autism. Journal of chemical neuroanatomy, 132, 102317. https://doi.org/10.1016/j.jchemneu.2023.102317