The body produce oxygen free radicals through the enzyme system and non-enzyme system, which can attack unsaturated fatty acid on biofilm and lead to lipid peroxidation and form lipid peroxide, such as aldehyde group (MDA), keto-, hydroxyl, carbonyl, etc. Oxygen free radicals cause cell damage not only by peroxidation of polyunsaturated fatty acids in biofilm, but also by decomposition products of lipid hydroperoxide. Detection of the MDA content can reflect the level of lipid peroxidation in cells and reflect level of cellular damage indirectly.

MDA in the catabolite of lipid peroxide can react with thiobarbituric acid (TBA) and produce red compound, which has a maximum absorption peak at 532 nm.

1.  It is recommended to fasten the glass tube mouth with preservative film and make a small hole in the film.

2.  Water-bath temperature (95-100℃) and incubation time (40 min) should be stabilized. Cool the tubes with running water immediately once the incubation finished.

3.  The supernatant for assay should not contain sediment, otherwise it will affect the OD values. It is recommended to use a pipette to take the supernatant.

4.   Accurately take 250 μL reaction solution into the 96 wells microplate and without bubble.

注：A：blank well，B：standard wells S1-S92：sample wells.

1.   Sample pretreatment

Collect the cells into a centrifuge tube, add 0.5 mL of reagent 5 working solution, mix fully for 2 min, then treat the cell with sonication or homogenization. Meanwhile, determine the protein concentration of supernatant (E-BC-K318-M, E-BC-K168-S, E-BC-K165-S).

2.       The measurement of samples

(1)   Blank tube: Take 0.1 mL of absolute ethyl alcohol (self-prepared) to the 1.5 mL EP tubes

Standard tube: Take 0.1 mL of 10 nmol/mL standard to the 1.5 mL EP tubes.

Sample tube: Take 0.1 mL of sample to the 1.5 mL EP tubes.

(2)   Add 1 mL of working solution into the wells of Step 1.

(3)   Mix fully with a vortex mixer. Tighten the tubes with preservative film and make a hole in the film. Incubate the tubes in 100℃ water bath for 40 min.

(4)   Cool the tubes to room temperature with running water. Centrifuge at 1078 g for 10 min.

(5)   Take 250 μL of supernatant to microplate and measure the OD value at 532 nm.