Mitoscreen

Analysis of mitochondrial membrane potential was conducted using the BD MitoScreen (BD Biosciences). To determine the amount of cells undergoing mitochondrial membrane potential (∆ψ_m) in response to cisplatin, the cells were incubated in JC-1 stain (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) for 15 min at 37°C in the dark, and then were washed two times with 1X assay buffer. Analysis on the ∆ψ_m was conducted based on the intensity of the fluorescence detected in both the FL-1 and FL-2 channels by using a flow cytometer (BD FACSCalibur; BD Biosciences).

Mitochondrial membrane potential was determined using a JC-1 assay kit (BD Mitoscreen, Cat No: 551302, San Diego, CA, USA). JC-1 remains as a monomer and emits green fluorescence at a low level of mitochondrial membrane potential, but the state of high mitochondrial membrane potential it forms aggregates and emits red fluorescence (Xuan et al., 2014 ).
A total of 75 × 10⁴ cells were cultured in T-25 flasks for 24 h. These were then treated with 370 and 666 μg/mL concentrations of DEM for 72 h and harvested. Cell suspensions were centrifuged for 5 min at 300g at room temperature. The supernatant part was removed, and 500 μL JC-1 Working Solution was added to the cell pellets and incubated for 15 min at 37 °C. At the end of that time the mixture was centrifuged at 400g for 5 min. The supernatants were then aspirated, and cell pellets were washed twice with 1 × Assay Buffer. All procedures were carried out according to the manufacturer’s recommendations. Data from 10⁴ cells were collected and analyzed on a flow cytometer (FACSCalibur, Becton Dickinson, East Rutherford, NJ, USA). The results were obtained from the ratio of red to green fluorescence and were expressed as relative mitochondrial membrane potential compared to negative control samples (cells with no test compound).

Mitochondrial membrane
potential was analyzed using the JC1 kit (BD MitoScreen, BD Biosciences)
following the manufacturer’s protocol.