LNCaP or PC3M cells were seeded on tissue culture dishes with cover glass bottom (FluoroDish, FD35, World Presicion Instruments, Inc.). Changes in mitochondrial membrane potential (ΔΨ) were imaged by means of the fluorescent ΔΨ-dependent lipophilic cationic dye tetramethyl-rhodamine ethyl ester (TMRE) which accumulates in active mitochondria. LNCaP and PC3M cells were stained with TMRE (0.1 µM, 10 min), treated with vehicle, CQ (20 μM) or FQ (20 μM) for 2 h and changes in intensity of TMRE signal were analyzed by confocal microscopy. To confirm the mitochondrial origin of the TMRE signal, at the end of the imaging protocol the cells were exposed to 5 µmol/L FCCP. TMRE fluorescence was excited by the 543 nm line of a 5 mW HeNe ion laser and the emitted fluorescence signal was captured at wavelengths above 560 nm.
Alternatively, ΔΨ was assessed using another ΔΨ-sensitive probe 3,3′-Dihexyloxacarbocyanine Iodide (DiOC6(3)) (318426, Aldrich). LNCaP or PC3M cells were treated with vehicle, CQ (15 μM) or FQ (15 μM) for 15 h, stained with DiOC6(3) (40 nM) for 15 min at 37 °C and analyzed by flow cytometry using Cyan LX9 cytometer (Beckman Coulter). Data analysis was performed using Summit 4.3 software (Beckman Coulter).
Alternatively, ΔΨ was assessed using another ΔΨ-sensitive probe 3,3′-Dihexyloxacarbocyanine Iodide (DiOC6(3)) (318426, Aldrich). LNCaP or PC3M cells were treated with vehicle, CQ (15 μM) or FQ (15 μM) for 15 h, stained with DiOC6(3) (40 nM) for 15 min at 37 °C and analyzed by flow cytometry using Cyan LX9 cytometer (Beckman Coulter). Data analysis was performed using Summit 4.3 software (Beckman Coulter).