Application

This kit can be used to measure the non-esterified free fatty acids (NEFA) content in animal blood, tissue and cells samples. 

Detection significance

NEFA is not only the product of fat hydrolysis, but also the substrate of fat synthesis. The concentration of NEFA is related to lipid metabolism, glucose metabolism and endocrine function.

Detection principle

Under the condition of weak acidity, non-esterified free fatty acids (NEFA) react with nantokite to form copper soap, which has a specific absorption peak at 715nm. The content of NEFA can be calculated by measuring the OD value at 715 nm. 

Experimental instrument

Micropipette, Vortex mixer, Magnetic stirrer, Centrifuge, Spectrophotometry (715 nm), Electronic analytical balance

Pretreatment of sample

[Note]

a) It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment. Bring all the reagents to room temperature before experiment.

b) The samples should be fresh collected and detect within 24 hours.

1. For blood sample:

Collect the blood sample and stand at room temperature for 1 hour, centrifuge at 2000 g for 15 min at 4℃., then take 0.2 mL of the supernatant. It is recommended to add 2.4 mL of Reagent 1, then oscillate at 4℃ for 3 hours to extract the NEFA. Centrifuge the sample at 2000 g for 10 min at 4℃. and take the supernatant for detection.

2. For tissue sample:

Wash the tissue with PBS (2~8℃, 0.01M, pH=7.4), then remove the water on surface of the tissue with absorbent paper. Weigh and mince the tissue, then add Reagent 1 according to the ratio of Weight (g): Reagent 1(mL) =1: 12, then oscillate at 4℃ for 3 hours to extract the NEFA. Centrifuge the sample at 2000 g for 10 min at 4℃ and take the supernatant for detection.

3. For cell samples:

Add Reagent 1 according to the ratio of cell number (106): Reagent 1 (mL)= 5: 1.2 (it is recommende to take 5×106 cells), then treat the sample with ultrasonic for 3 min on ice (200 W, 2 seconds/time, interval for 3 seconds), then oscillate at 4℃ for 3 hours to extract the NEFA. Centrifuge the sample at 2000 g for 10 min at 4℃, and take the supernatant for detection.

Operation procedure

1. Dilution of standard

Dilute 10 mmol/L standard solution with reagent 1 to a serial concentration. The recommended dilution gradient is as follows: 2.0, 1.5, 1.0, 0.5, 0.25, 0.1, 0 mmol/L.

2. Operation steps

1) Standard rube: Add 1 mL of standard with different concentrations and add 0.5 mL of Reagent 4.

Control tube: Take 1 mL of the supernatant and add 0.5 mL of Reagent 3. 

Sample tube: Take 1 mL of the supernatant and add 0.5 mL of Reagent 4. 

2) Oscillate for 5 min and stand at room temperature for 5 min.

3) Set spectrophotometry to zero with reagent 1 and take 0.8 mL of the upper layer liquid into 1 cm cuvette and measure the OD value at 715 nm.

Note: It can be refer to the following operating table

Technical parameters

1. The sensitivity of the kit is 0.05 mmol/L.

2. The intra-assay CV is 2.2 % and the inter-assay CV is 6.1 %.

3. The recovery of the kit is 100 %.

4. The linear range of the kit is 0.05-2 mmol/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.