2.7. Mitochondrial Respiratory Chain Complexes Activity Assay

L02 cells were cultured in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in a 5% CO₂ incubator at 37 °C. L02 cells in logarithmic phase were collected, and 2 × 10⁵ cells/well were inoculated in 6-well plates. Cells were incubated at 37 °C until the cell density reached 80%, then treated with culture medium and emodin at 50 μΜ for 48 h. After treatment the cells were collected, centrifuged, and the cell pellet was retained. An appropriate amount of lysis buffer was added for suspended cell, and then the mitochondria were extracted according to the instructions, and 5 × 10⁷ cells per time. After that, the activities of mitochondrial respiratory chain complexes I, II, III, IV, and V were quantitatively detected through the colorimetric method; the control group and the administered group samples were prepared in triplicate, respectively.

The activity values of the complexes are calculated as follows:

Activity value = [(test sample OD value − background OD value) × system volume × test sample dilution multiple]/[test sample volume × X (millimole absorptivity) × reaction time(min) × test sample protein concentration (mg prot/mL)]

Different complexes have different X values, these being 5.5, 21.8, 21.84, 21.84, and 6.22 for complexes I, II, III, IV, and V, respectively. Prot is an abbreviation of protein.