Nitric Oxide Assay Kit, Fluorometric

Assay kit useful for the rapid quantitative measurement of nitric oxide (NO). Displays 50-fold increased sensitivity over the colorimetric nitric oxide assay kit (Cat. No. 482650). The assay is based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by the addition of 2,3-diaminonapthalene (DAN), and NaOH, which converts nitrite to a fluorescent compound. Fluorescence measurements of this compound accurately determine the nitrite (NO₂^-) concentration (excitation max.: 365 nm; emission max.: 450 nm).Do not use with nitrate- or nitrite-containing tissue culture media such as RPMI.

Assay Buffer, Nitrate Reductase, Enzyme Cofactors, Nitrate Standard, Nitrite Standard, DAN Reagent, NaOH, Microtiter Plates, Plate Covers, and a user protocol.

Toxicity: Multiple Toxicity Values, refer to MSDS (O)

Assay Time: 3-5 h

1. Assay Buffer: Dilute the contents of the vial to 100 ml with HPLC-Grade water. Use this buffer for diluting samples, as needed, prior to assay. Store at 4°C. This buffer will be stable for ~2 months at 4°C.2. Nitrate Reductase: Reconstitute the contents of vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store at -20°C. Allow only one time freezing and thawing of this solution.3. Enzyme Co-factors: Reconstitute the contents of this vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store at -20°C. Allow only one time freezing and thawing of this solution.4. Nitrate Standard: Remove the vial stopper slowly to minimize disturbance of the lyophilized powder. Reconstitute the lyophilized nitrate standard using 1.0 ml of Assay Buffer. The concentration of this solution is 2 mM. Swirl to ensure that powder clinging to the sides of the vial is dissolved. Vortex gently. Store all stock solutions at 4°C; do not freeze after reconstitution. When stored under these conditions, the nitrate standard is stable for at least three months.5. Nitrite Standard: Remove the vial stopper slowly to minimize disturbance of the lyophilized powder. Reconstitute the lyophilized nitrite standard using 1 ml of Assay Buffer. The concentration of this solution is 2 mM. Swirl to ensure that powder clinging to the sides of the vial is dissolved. Vortex gently. Store all stock solutions at 4°C; do not freeze after reconstitution. When stored under these conditions, the nitrite standard is stable for at least three months.6. Fluorometric reagent (DAN): Ready to use. Store at 4°C. Do not add water or assay buffer to this vial. 7. NaOH : Ready to use. Store at 4°C. Do not add water or assay buffer to this vial.

1. Culture Medium: Culture medium such as RPMI 1640 may contain high levels of nitrate. It is best not to use these types of media, particularly when small changes in nitrate levels are measured. If it is absolutely necessary to use this type of medium then cellular nitrate/nitrite levels can be quantitated by subtracting the level of nitrate/nitrite in the medium (in the absence of cells) from the total levels. Phenol red and fetal bovine serum (FBS) added to the medium can cause a significant reduction in fluorescence. Whenever possible these components should be avoided. The effect of media components on fluorescence intensity must be assessed by making the nitrate or nitrite standard curve in the presence of an equivalent amount of the phenol red or FBS. To obtain maximum signal response, it is best to use 10 or 20 µl sample volumes. Use of larger sample volumes (30 to 50% of the final reaction volume) can lead to quenching of fluorescence. To prepare a standard curve in the presence of media, simply prepare the nitrate or nitrite standard curve substituting the amount of media desired in the place of assay buffer. For the measurement of nitrate plus nitrite an incubation period of 1 h is required for the reaction to reach completion.2. Plasma or Serum: Ultrafilter plasma or serum samples through a 10 or 30 kDa cut-off filter using a commercially available centrifuge or microfuge ultrafiltration device. This procedure removes hemoglobin thereby avoiding the reduction in fluorescence intensity. Assay for nitrate and/or nitrite using a maximum of 10 µl filtrate. The conversion of nitrate to nitrite requires 1-2 h (for ≥95% conversion).3. Tissue Homogenates: Homogenize the sample in phosphate-buffered saline (PBS, pH 7.4) and centrifuge at 10,000 x g for 20 min. Centrifuge at 100,000 x g for 30 min (this second centrifugation is optional, but will increase filtration rates). Ultrafilter the supernatant through a 10 or 30 kDa cut-off filter. Use 10 µl of the filtrate for nitrate and/or nitrite assay. The conversion of nitrate to nitrite requires about 2 h for ≥95% conversion.

Upon arrival, store the entire contents of the kit at -20°C until use. For storage information on individual components following initial thawing and reconstitution, please consult the section on Pre-Assay Preparation.

Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.

Miles, A.M., et al. 1996. Methods Enzymol. 268, 105.
Misko, T.P., et al. 1993. Anal. Biochem. 214, 11.
Green, L.C., et al. 1982. Anal. Biochem.126, 131.

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