Lsm 880 live confocal laser microscope

JC-1 (5, 59, 6, 69-tetrachloro-1, 19, 3, 39-tetraethylbenzimidazolcarbocyanine iodide) is a cationic dye used to target mitochondria. When a cell is healthy, JC-1 aggregated inside the mitochondria emit red fluorescence (590 ± 10 nm). However, when a cell is unhealthy, the mitochondrial potential is low, and the JC-1 monomers stay in the cytoplasm and emit green fluorescence (525 ± 10 nm). To assess the mitochondrial membrane potential of the DTOS emulsion, HeLa cells were seeded in confocal dishes (µ-Slide 8 Well; ibidi, Fitchburg, WI, USA) (8 × 10³ cells/well) and allowed to adhere at 37 °C under 5% CO₂ for 24 h. Sample-treated (24 or 48 h) cells were then washed and incubated with 2 μM of JC-1 at 37 °C for 20 min. Cells were washed twice with DPBS, and analyzed using a Zeiss LSM 880 Live confocal laser microscope (Zeiss, Oberkochen, Germany).

HeLa cells (8 × 10³ cells/well) were seeded in confocal dishes (µ-Slide 8 Well; ibidi, Fitchburg, WI, USA) and allowed to adhere in 270 µL of DMEM media (10% FBS and 1% (w/v) penicillin/streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂ for 24 h. Then, cells were incubated with 30 µL of Nile red emulsion for 24 h. The medium was removed, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) twice, and the nucleus was stained for 10 min with 200 µL of bisbenzimide (Hoechst 33342). The mitochondria were stained with 200 µL of MitoGreen (2 µM) for 20 min. Finally, cells were washed and observed using a Zeiss LSM 880 Live confocal laser microscope (Zeiss, Oberkochen, Germany).