Application

This kit can measure NO content of all kinds of serum, plasma, culture cells, culture supernatant, tissue and other samples. 

Detection significance

NO is a kind of highly reactive free radical, which has the function of the second messenger and neurotransmitter, and it is also a kind of effector molecule, which has a wide range of physiological functions in vivo, such as relax vascular smooth muscle, regulate cerebral blood flow, mediate cytotoxic effect and immune regulation, participate in learning and memory, etc. Half life of NO is very short. NO in blood is mainly produced by vascular endothelial cells, vascular smooth muscle cells, platelets, macrophages and so on. It exists in the form of nitrate and nitrite, and the concentration of NO can calculate indirectly by the concentration of nitrate and nitrite.

Detection principle

No is easy to oxidize in vivo or in aqueous solution to form NO2-, and react with chromogenic agent to produce reddish azo compound. There is a linear relationship between the concentration of azo compound and the concentration of NO. The NO content can be calculated indirectly by measuring the OD value at 550 nm. The action of reagent 1 and reagent 2 is to remove the interference of colored matter in sample.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, 37℃ incubator, Spectrophotometer (550 nm)

Sample preparation

1.   Cells: Collecting cell sediment, add 1 mL PBS (0.01M, PH7.4) to suspend the cells, then homogenize the cells in ice bath, centrifuge at 3100 g for 10min, collect the supernatant for test. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

2.   Serum (plasma) or culture supernatant: Detect the sample directly.

3.   10% homogenate: Weigh the tissue accurately and add PBS (0.01M, PH7.4) at a ratio of weight (g): volume (mL) =1:9, homogenize the tissue in ice bath, centrifuge at 3100 g for 10 min, collect the supernatant for test. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

Mix fully and stand for 20 min. Set to zero with distilled water and measure the OD values of each tube at 550 nm with 1 cm diameter cuvette.

Note: a* = Sample volume= Standard volume = Distilled water volume.

Technical parameter

1.  The sensitivity of the kit is 0.97 μmol/L.

2.  The intra-CV is 3.4% and the inter CV is 5.2%.

3.  The recovery of the kit is 99%.

4.  The detection range of the kit is 0.97-700 μmol/L.

Notes：

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.

5. It is recommended to use a disposable plastic tube or glass tube must be washed clean.

6. Hemolysis and turbid serum have an effect on the results of the experiment.

7. Serum samples can be stored for 3 days at 4℃ and for a month at -20℃.