Mitochondrial Membrane Potential Assay

The MCF-7 cells were treated with compounds 9 and 13 and doxorubicin, and the integrity of MMP was analyzed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Germany) staining as previously reported [27 (link)–29 (link)]. Cells were tested in 6-well plates (3 mL, 1 × 10⁵ cells/mL) and the incubation time was 72 h in humidified 5% CO₂ atmosphere at 37 °C. The tested concentrations were ¼ × IC₅₀, ½ × IC₅₀ and IC₅₀. Untreated cells (control) were used for comparison with treated cells. JC-1 staining was performed according to the manufacturer’s protocol as reported previously [23 (link)]. Cells were then measured in a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). The JC-1 signal was measured at an excitation wavelength of 561 nm (150 mW) and detected using a 586/15 nm band-pass filter. The signal was analyzed at an excitation wavelength of 640 nm (40 mW) and detected using a 730/45 nm bandpass filter. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson). All experiments were performed at least in triplicates.