Nitric oxide (NO) is a reactive radical that plays an important role in many key physiological functions. NO, an oxidation product of arginine by nitric oxide synthase, is involved in host defense and development, activation of regulatory proteins and direct covalent interaction with functional biomolecules. Simple, direct and automation-ready procedures for measuring NO are becoming popular in Research and Drug Discovery. Since NO is oxidized to nitrite and nitrate, it is common practice to quantitate total NO2 -/NO3 - as a measure for NO level. BioAssay Systems' QuantiChromTM Nitric Oxide Assay Kit is designed to accurately measure NO production following reduction of nitrate to nitrite using improved Griess method. The procedure is simple and the time required for sample pretreatment and assay is reduced to only 40 min.

KEY FEATURES

Sensitive and accurate. Detection range 0.1 - 50 μM in 96-well plate.

Rapid and reliable. Using optimized Cd/Cu reagent, the time required for reduction of NO3 - to NO2 - is 15 min at >98.5% conversion rate.

Simple and high-throughput. The procedure involves mixing sample with two reagents, incubation for 5 min and reading the optical density. Can be readily automated to measure thousands of samples per day.

APPLICATIONS:

Direct Assays: NO in plasma, serum, urine, tissue/cells and foods.

Drug Discovery/Pharmacology: effects of drugs on NO metabolism.

PROCEDURES

Sample treatment: tissue or cell samples are homogenized in 1 x PBS (pH 7.4). Centrifuge at 10,000g or higher at 4°C. Use supernatant for NO assay. Samples that need deproteination include serum, plasma, whole blood, cell culture media containing FBS, tissue or cell lysates. Urine and saliva do not need deproteination.

Reagent preparation: dilute 20x ZnSO4 and 30 x NaOH to 1-fold with water. The final concentrations are 75 mM ZnSO4 and 55 mM NaOH, respectively. Dilute Activation Buffer by mixing 1 volume of 3 x Activation Buffer with 2 volumes of distilled water. All diluted solutions can be stored at 4°C for 12 months.

1. Standards. Prepare 600 μL 50 μM Premix by mixing 30 μL 1.0 mM Standard and 570 μL distilled water. Dilute standard in 1.5-mL centrifuge tubes as shown in the Table. Transfer 100 μL diluted standards into wells of a clear-bottom 96-well plate.

2. Deproteination. Mix 100 μL sample with 80 μL 75 mM ZnSO4 in 1.5- mL tubes. If precipitation occurs, centrifuge 5 min at 14,000 rpm. Transfer supernatant to a clean tube containing 120 μL 55 mM NaOH. Pellet protein precipitates again (dilution factor n = 3). Transfer 210 μL supernatant and mix with 70 μL Glycine Buffer in a 1.5-mL centrifuge tube. If solution remains clear in these steps, deproteination is not required. Directly transfer 210 μL sample (dilution factor n = 1) and mix with 70 μL Glycine Buffer in a 1.5-mL centrifuge tube.

3. Activation of Cd. The number of Cd granules to be used is 3 x the number of samples. Transfer Cd granules in a 50-mL centrifuge tube. Wash Cd three times with water. Remove residual water with a pipet. Add 200 μL diluted 1 x Activation Buffer per granule and incubate 5 min at room temperature. Swirl tube intermittently. Wash three times with water. Activated Cd should be used within 20 min.

Note: cadmium is a toxic and expensive metal. Avoid direct contact (wear gloves). 15 grams of Cd granules are provided which is sufficient for about 50 samples. Used Cd granules can be washed and stored in 0.1 N HCl. Prior to assay, wash Cd granules 3 times with water and regenerated using the activation procedure. Cd granules can be regenerated and used 7 times without loss of activity.

4. Nitrate Reduction. Dry the activated Cd granules on a filter paper (e.g. Kimwipes® EX-L). Add three Cd granules per sample (Step 2) and shake tubes intermittently. Incubate 15 min at room temperature. Transfer 2 x 100 μL samples (duplicate) into wells of the 96-well plate.

5. Assay. Add 50 μL Reagent A to all wells and tap plate lightly to mix. Add 50 μL Reagent B and mix. Incubate 5 min at room temperature. Read OD at 500-570nm (peak 540 nm).

Prepare standards and samples as described for the 96-well procedure. After the reduction step, mix 500 μL diluted standard and deproteinated sample with 250 μL Reagent A and 250 μL Reagent B. Measure OD540nm in the cuvet.

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The NO concentration of Sample is calculated as

ODSAMPLE and ODBLANK are optical density values of the sample and water, respectively. n is the dilution factor (see Step 2). Conversions: 1 mg/dL NO equals 333 μM, 0.001% or 10 ppm.

Pipeting devices, centrifuge tubes and table centrifuge.

Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Cuvets and spectrophotometer for measuring optical density at 540 nm.

Antioxidants and nucleophiles (e.g. b-mercaptoethanol, glutathione, dithiothreitol and cysteine) may interfere with this assay. Avoid using these compounds during sample preparation.

Rat serum and fresh human urine was analyzed using the 96-well protocol. The NO levels were 28.7 ± 0.3 and 6.4 ± 0.2 μM, respectively. Coefficient of Variance < 4%. Day-to-day variation < 8%.

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