The half-life of NO is extremely short, it exists in form of nitrate or nitrite produced by vascular endothelial cell, vascular smooth muscle cell, platelet, and macrophage and so on. The concentration of NO can be indirectly measured by detecting that of nitrate or nitrite.
NO react with oxygen and water to generate nitrate or nitrite which can form a kind of pale red azo compound when meet with nitrate chromogenic reagent, the absorbance of the compound can be measured to calculate the concentration of NO indirectly.
1. Use disposable EP tubes or clean glass tubes with stopper for centrifugation.
2. The supernatant for assay should not contain sediment, otherwise it will affect the results.
3. Samples can be stored at -20 ℃ for 1-2 months, the lower the temperature, the longer the storage time.
4. All reagents should be prepared the day before the assay, let it fully dissolved. Please add reagents to the bottom of well vertically and slowly, avoid to add on the wall of well and generate bubble.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | A | A | S1 | S9 | S17 | S25 | S33 | S41 | S49 | S57 | S65 | S73 |
B | B | B | S2 | S10 | S18 | S26 | S34 | S42 | S50 | S58 | S66 | S74 |
C | C | C | S3 | S11 | S19 | S27 | S35 | S43 | S51 | S59 | S67 | S75 |
D | D | D | S4 | S12 | S20 | S28 | S36 | S44 | S52 | S60 | S68 | S76 |
E | E | E | S5 | S13 | S21 | S29 | S37 | S45 | S53 | S61 | S69 | S77 |
F | F | F | S6 | S14 | S22 | S30 | S38 | S46 | S54 | S62 | S70 | S78 |
G | G | G | S7 | S15 | S23 | S31 | S39 | S47 | S55 | S63 | S71 | S79 |
H | H | H | S8 | S16 | S24 | S32 | S40 | S48 | S56 | S64 | S72 | S80 |
Note: A: blank wells; B-H: standard wells; S1-S80: sample wells
1. Pre-treatment
| Standard tube | Sample tube |
Different concentrations of sodium nitrite (μL) | A* |
|
Sample (μL) |
| A* |
Reagent 1 (μL) | 200 | 200 |
Reagent 2 (μL) | 100 | 100 |
Mix fully and stand for 15 min at room temperature, centrifuge it at 3100 g for 10 min, take 160 μL of the supernatant for the following procedure. | ||
Note: a: For serum or plasma samples, A* is 200-300 μL.
b: For tissue or cell homogenates, A* is 100-300 μL.
2. Reaction
| Standard tube | Sample tube |
Supernatant (μL) | 160 | 160 |
Chromogenic reagent (μL) | 80 | 80 |
Mix thoroughly for 2 min, stand for 15 min at room temperature, measure the OD of each well with micro-plate reader immediately at 550 nm wavelength. | ||
| Detection range | 0.16-300 μmol/L | Average inter-assay CV | 3.7% |
| Sensitivity | 0.16 μmol/L | Average intra-assay CV | 2.4% |
| Average recovery rate | 102% |
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