Mitotracker green fm

For cell imaging studies, cells were seeded in 8 well chamber slides at density 20,000 cells/well, differentiated and treated with 6-OHDA in the absence or presence of TEMPOL. After 24 h, drugs and media were removed and 200 μL 5μM MitoSOX Red (diluted in warm Hank’s Balanced Salt Solution (HBSS); Thermo Fisher Scientific, Scoresby, Australia) was added and the mixture incubated for 30 min in a humidified chamber. The wells were then washed with warm HBSS before addition of 200 μL 10 nM MitoTracker Green FM (in HBSS; Thermo Fisher Scientific, Scoresby, Australia) for 10 min in a humidified chamber. After washing with HBSS, chamber slides were imaged using a Zeiss Axio Scope.A1 (Zeiss, Sydney, Australia) with excitation/emission of 510 nm/580 nm for MitoSOX Red and 490 nm/516 nm for MitoTracker Green FM. For quantification, cells seeded in a 96-well plate at 8000 cells/well were differentiated then treated with 6-OHDA ± TEMPOL for 24 h, before addition of 200 μL of 5μM MitoSOX Red. Finally, fluorescence was determined at 510 nm/580 nm (excitation/emission) using a TECAN M200 PRO plate reader (Tecan, Melbourne, Australia).

Expression and/or localization of AIF, γH2AX and PINK1 were investigated by immunofluorescence microscopy. In brief, approximately 6-8000 cervical cancer cells were seeded on 8-chambered slides, and treated with SHetA2 or vehicle for 24 hours. Then the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX‐100 in PBS. Following blocking with 4% BSA in PBS, cells were incubated with and MitoTracker™ Red CMXRos (#M7512, Thermo Fisher Scientific) for 45 minutes and then by primary antibodies for AIF (#MA5-15880, Thermo Fisher Scientific) or γH2AX or PINK1 at 1:100 dilutions in 1% BSA‐PBS for overnight. After washing, cells were stained with Alexa Fluor 488‐labeled secondary antibody for 1 hour. DAPI (blue) was used to stain the nucleus. Cell images were acquired with a 63X objective using a Zeiss Axio Observer Z1 (Göttingen, Germany). For mitophagy detection, cervical cancer cells treated with SHetA2 or DMSO were stained with LysoTracker™ Deep Red (#L12492, Thermo Fisher Scientific) and MitoTracker™ Green FM, and live-cell imaging was performed with a 63X objective using a Zeiss Axio Observer Z1 (Göttingen).

Live HAECs exposed to either static (STT) or LSS were incubated with 200 nM pre-warmed MitoTracker Green FM or MitoTracker Red CMXRos (Molecular Probes) solution at 37°C for 30 min. After removal of the incubation solution, cells were washed three times with pre-warmed PBS and then mounted in Hank's balanced salt solution. For quantitative analyses, more than 100 images per each group were acquired using an epi-fluorescence upright microscope with a 63x objective oil lens. For MitoTracker Green FM staining, excitation/emission wavelengths were set at 470/525 nm (FL filter Set 38, Zeiss), and for MitoTracker Red CMXRos staining, excitation/emission wavelengths were set at 587/647 nm (FL filter Set 64HE). Images were initially acquired using an AxioCam MRm and AxioVision image processing system (Zeiss), and the fluorescence intensities were assessed using Image J software (NIH).