Ci enzyme activity microplate assay kit

The analysis of CI enzyme activity in sperm and testes was performed using a CI Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, UK) following the kit protocol. In short, CI was extracted and introduced into a microplate. After incubation at room temperature for 3 h, the microplate was washed, and assay solution was added. Absorbances of samples were tested at 450 nm for 30 min with an interval of 20 s, following the kinetic program in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, USA). Activity was expressed as the change in absorbance per minute per amount of sample loaded into a well. The data are shown as the standardized ratio of the activity in the ORN-treated rats to that in the control animals.

CI activity was determined by measuring the oxidation of NADH to NAD⁺ and the simultaneous reduction of the provided dye (ε = 25.9/mM per well), which led to increased absorbance at Optical density (OD) 450 nm. CI activity was quantified using a CI Enzyme Activity Microplate Assay Kit (Abcam) per the manufacturer’s instruction and calculated as mOD/min per µg protein.