Lipid peroxidation, as an indicator of oxidative stress in cell and tissue, has been identified as a kind of cellular damage. Lipid peroxide is unstable and can decompose into complex mixture including carbonyl compounds. Polyunsaturated fatty acid peroxide is decomposed into Malondialdehyde (MDA) and 4- hydroxyl olefins (HAE). Detection of LPO, MDA and HAE has been an indicator of lipid peroxidation.

With 45℃ incubation for 60 min, one molecule of LPO react with two molecule of chromogenic reagent, to produce a stable chromophore which have the maximum absorption peak at 586 nm. The content of LPO in samples can be calculated by standard curve or calculation formula.

1.  The EP tube needs to be sealed to avoid leakage.

2.  The supernatant added to 96-well microplate must be clarified, otherwise centrifuge again.

3.  Please carry out the experiment in the fume hood and wear disposable gloves, prevent the reagents splashing dashed into eyes and skin.

Dilute 100 μmol/L standard solution with absolute ethanol to a serial concentration. The recommended dilution gradient is as follows: 80, 50, 40, 30, 20, 10, 5, 0 μmol/L.

1)    Standard well: add 200 μL of standard solution with different concentrations into the 1.5 mL EP tube.

Sample well: add 200 μL of Sample into the 1.5 mL EP tube.  

2)    Add 650 μL of chromogenic agent, cover the caps and mix fully.

3)    Add 150 μL of Reagent 3, cover the caps and mix fully.

4)    Incubate at 45℃ for 60 min. Cool to room temperature with running water.

5)    Centrifuge at 1100 g for 10 min. Take 200 μL of supernatant to 96-well microplate, measure the OD values of each well at 586 nm with Microplate reader.