Griess Assay for Nitrite Quantification (Indirect Nitric Oxide Measurement)

A colorimetric microplate assay that quantifies nitrite (NO2-), the stable end-metabolite of nitric oxide (NO), in cell-free culture supernatant. Under acidic conditions nitrite diazotizes sulfanilic acid; the diazonium salt then couples with N-(1-naphthyl)ethylenediamine (NEDA) to form a pink azo dye read near 540-548 nm. Concentrations are interpolated from a matrix-matched sodium nitrite (NaNO2) standard curve (0-100 uM) with a medium-only blank. Used as an index of NO production in stimulated cells (e.g., LPS-activated macrophages).

biochemistry Beginner 45 min 8 steps

Stage 01 / 05Preparation

Prepare the Griess reagents and nitrite stock

Acidified sulfanilic acid and NEDA are the two Griess reagents. A sodium nitrite stock supplies the working standard.

Done
STEP 1Prepare the two Griess reagents15 min
Prepare 1% (w/v) sulfanilic acid: dissolve 0.5 g sulfanilic acid in 50 mL of 2.5% (v/v) phosphoric acid. Prepare 0.1% (w/v) NEDA: dissolve 0.05 g NEDA in 50 mL ddH2O. Transfer both reagents to amber or foil-wrapped bottles and keep protected from light until use.

WhyAcidified sulfanilic acid forms the diazonium intermediate and NEDA is the coupling amine; these two solutions are the complete Griess reagent set.
CautionNEDA and the working solutions are light-sensitive and degrade over time. Keep them dark and use promptly; do not store diluted reagents long-term at room temperature.
Done
STEP 2Prepare the NaNO2 stock and intermediate5 min
Prepare a 1 M NaNO2 stock: dissolve 0.069 g NaNO2 in 1 mL ddH2O. Dilute the 1 M stock 1:100 with ddH2O to obtain 10 mM NaNO2. Dilute the 10 mM solution 1:100 with culture medium to obtain a 100 uM working standard.

WhyNitrite is the stable, measurable oxidation product of NO; a defined NaNO2 series is the calibrator that converts absorbance to concentration.

Stage 02 / 05Loading

Load matrix-matched standards and supernatant

Nitrite accumulated in cell-free supernatant represents nitric oxide production by stimulated cells. Standards and the blank use the same culture medium as the samples.

Done
STEP 3Build the nitrite standard curve10 min
From the 100 uM working standard, prepare standards in culture medium at 0, 6.25, 12.5, 25, 50, and 100 uM. Use neat culture medium as the 0 uM blank. Dispense 100 uL of each standard into wells in duplicate or triplicate.

WhyStandards prepared in the same medium as the samples (matrix matching) correct for medium absorbance and matrix effects, giving an accurate calibration.
CautionPrepare the curve in culture medium, not ddH2O . A water-based curve will misquantify medium-based samples.
Done
STEP 4Load the samples5 min
Dispense 100 uL of each cell-free supernatant into wells in duplicate or triplicate. Include a medium-only blank that matches the sample medium.

WhyReplicate wells define assay precision and the matched blank sets the zero baseline for subtraction.

Stage 03 / 05Diazotization

Diazotize nitrite with sulfanilic acid

Under acidic conditions, nitrite converts sulfanilic acid into a diazonium salt. A fixed five-minute interval keeps the first reaction step comparable across wells.

Done
STEP 5Add sulfanilic acid and run diazotization5 min
Add 50 uL of 1% sulfanilic acid to every well (standards and samples). Mix gently and incubate at room temperature for 5 min, protected from light.

WhyUnder acidic conditions nitrite converts sulfanilic acid into a diazonium salt; this is the first, rate-defining step of the Griess reaction.

Stage 04 / 05Coupling

Add NEDA to develop the azo dye

NEDA couples with the diazonium salt to form the pink azo dye. Its absorbance tracks nitrite concentration.

Done
STEP 6Add NEDA to develop color5 min
Add 50 uL of 0.1% NEDA to every well. Mix gently and let the pink azo dye develop at room temperature (~5-10 min), protected from light.

WhyNEDA couples to the diazonium salt to form the pink azo dye whose absorbance is proportional to nitrite concentration.

Stage 05 / 05Readout

Read absorbance and interpolate nitrite

Read the developed plate near 548 nm, subtract the medium-only blank, then interpolate sample nitrite from the matrix-matched standard curve.

Done
STEP 7Read absorbance2 min
Read OD at 548 nm (acceptable 540-570 nm) on the microplate reader, using 570 nm as reference if available. Read all wells within a single, short time window after NEDA addition.

WhyThe azo dye absorbs maximally near 540-548 nm; reading on one fixed schedule keeps all wells at comparable development.
CautionDevelop and read all wells on the same timing. Staggered timing biases sample-to-sample comparison.
Done
STEP 8Calculate nitrite concentration5 min
Subtract the 0 uM blank OD from every well. Fit a linear regression of blank-corrected OD versus nitrite concentration (uM) from the standards; record R2. Interpolate each sample: C (uM) = (OD_sample - b) / m, where m is the slope and b is the intercept of the standard curve.

WhyNitrite accumulation in supernatant is the validated surrogate index of cellular NO production; interpolation against the matrix-matched curve converts OD into concentration.

Griess Assay for Nitrite Quantification (Indirect Nitric Oxide Measurement)

biochemistry · Beginner · 45 min

https://labgrimoire.com/protocols/griess-nitrite-assay/

Materials

☐ Sulfanilic acid: 0.5 g (Dissolve in 50 mL of 2.5% (v/v) phosphoric acid to make 1% (w/v) sulfanilic acid reagent; store in an amber/foil-wrapped bottle, protected from light.)
☐ Phosphoric acid (H3PO4), 2.5% (v/v): 50 mL (Acidifying diluent for the sulfanilic acid reagent; provides the acidic condition required for diazotization.)
☐ N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA, also "NED"): 0.05 g (Dissolve in 50 mL ddH2O to make 0.1% (w/v) NEDA reagent; light-sensitive, store protected from light and prepare fresh or store short-term at 4 C.)
☐ Sodium nitrite (NaNO2): 0.069 g (Dissolve in 1 mL ddH2O for a 1 M stock (MW 69.0 g/mol); primary standard for the calibration curve.)
☐ Culture medium (sample matrix): as needed (The same medium used for the experimental supernatant; used to prepare all standards and the 0 uM blank (matrix matching).)
☐ Deionized water (ddH2O): as needed (For the initial NaNO2 stock and 10 mM intermediate dilution only.)
☐ Test supernatant: 100 uL per well (Cell-free culture supernatant; assay fresh or store at -80 C until measurement.)

Equipment

☐ Microplate reader (ELISA reader) (Absorbance read at 548 nm (acceptable range 540-570 nm); use 570 nm as reference wavelength if available.)
☐ Non-coated, non-binding 96-well microplate (flat-bottom) (Avoid high protein-binding / tissue-culture-treated high-binding plates, which can distort readings.)
☐ Micropipettes and a multichannel pipette (For accurate 50-100 uL dispensing and parallel reagent addition.)
☐ Volumetric flasks / graduated tubes (For reagent and standard preparation.)
☐ Amber bottles or aluminum foil (To protect light-sensitive reagents during preparation and storage.)

Procedure

☐ Prepare the two Griess reagents (15 min)
Prepare 1% (w/v) sulfanilic acid: dissolve 0.5 g sulfanilic acid in 50 mL of 2.5% (v/v) phosphoric acid. Prepare 0.1% (w/v) NEDA: dissolve 0.05 g NEDA in 50 mL ddH2O. Transfer both reagents to amber or foil-wrapped bottles and keep protected from light until use.
☐ Prepare the NaNO2 stock and intermediate (5 min)
Prepare a 1 M NaNO2 stock: dissolve 0.069 g NaNO2 in 1 mL ddH2O. Dilute the 1 M stock 1:100 with ddH2O to obtain 10 mM NaNO2. Dilute the 10 mM solution 1:100 with culture medium to obtain a 100 uM working standard.
☐ Build the nitrite standard curve (10 min)
From the 100 uM working standard, prepare standards in culture medium at 0, 6.25, 12.5, 25, 50, and 100 uM. Use neat culture medium as the 0 uM blank. Dispense 100 uL of each standard into wells in duplicate or triplicate.
☐ Load the samples (5 min)
Dispense 100 uL of each cell-free supernatant into wells in duplicate or triplicate. Include a medium-only blank that matches the sample medium.
☐ Add sulfanilic acid and run diazotization (5 min)
Add 50 uL of 1% sulfanilic acid to every well (standards and samples). Mix gently and incubate at room temperature for 5 min, protected from light.
☐ Add NEDA to develop color (5 min)
Add 50 uL of 0.1% NEDA to every well. Mix gently and let the pink azo dye develop at room temperature (~5-10 min), protected from light.
☐ Read absorbance (2 min)
Read OD at 548 nm (acceptable 540-570 nm) on the microplate reader, using 570 nm as reference if available. Read all wells within a single, short time window after NEDA addition.
☐ Calculate nitrite concentration (5 min)
Subtract the 0 uM blank OD from every well. Fit a linear regression of blank-corrected OD versus nitrite concentration (uM) from the standards; record R2. Interpolate each sample: C (uM) = (OD_sample - b) / m, where m is the slope and b is the intercept of the standard curve.