Jc 10 assay kit

The mitochondria play an important role in apoptosis detection and induction of cell death [36 ]. The change in mitochondrial membrane potential (MMP) was measured using the JC-10 assay kit (Sigma-Aldrich, USA) according to vendor’s instructions. This uses a dual emission fluorescent dye capable of entering the mitochondria and emits either red or green fluorescence depending on the state of the mitochondrial membrane. Red fluorescence is seen in normal polarized mitochondria while a green fluorescence is obtained when the mitochondrial membrane potential decreases and the membrane is depolarized causing the dye to diffuse into the cytoplasm of the cell. Treated DU-145 cells (with docetaxel, varying concentrations of EAB, annonacin, control and 10 μM of H₂O₂) were incubated in 96-well plates for 72 h. After incubation, the media was removed, cells were washed with PBS then stained with 50 μL/well of the JC-10 dye loading solution. The plate was then incubated for 50 mins in a 37 °C incubator protected from light after which 50 μL/well of assay buffer B was added, then fluorescence intensity subsequently read at excitation/emission wavelengths of 490/525 nm and 540/590 nm [37 (link)]. Results are recorded as a ratio of red to green fluorescence.