Laser scanning microscope 5 pascal

Cells were treated with fucodiphlorethol G (20 μM), exposed to UVB radiation (30 mJ/cm²) 1 h later, and then incubated for another 12 h at 37°C. Cells were stained with JC-1 (15 μM). For image analysis, JC-1-stained cells were mounted in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope. In addition, JC-1-stained cells were analyzed by flow cytometry (Troiano et al., 2007 (link)).

Mitochondrial Ca²⁺ levels were monitored using Rhod-2 AM (Mészáros et al., 2012 (link)). Cells were treated with shikonin for 48 h; harvested, washed, resuspended in PBS containing 1 µM Rhod-2 AM; and incubated for 15 min at 37°C. Subsequently, the cells were washed and suspended in PBS for further analysis by flow cytometry. To confirm the flow cytometry results, the cells were seeded in 4-well chambers, and image analysis was conducted by loading cells with Rhod-2 AM for 30 min at 37°C. After washing, the stained cells were mounted on microscope slides with mounting medium (DAKO, Carpinteria, CA, USA). Images were captured on a confocal microscope using the Laser Scanning Microscope 5 PASCAL software (Carl Zeiss, Jena, Germany).

JC-1 was added to each well and cells were incubated for 30 min at 37°C. Stained cells were washed with phosphate-buffered saline (PBS), coverslips were mounted onto microscope slides in mounting medium (DAKO, Carpinteria, CA, USA), and slides were examined using a confocal microscope. Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) (Cossarizza et al., 1993 (link)). In addition, mitochondrial membrane potential was analyzed by flow cytometry. Cells were harvested, washed, suspended in PBS containing JC-1 (10 μg/ml), incubated for 30 min at 37°C, and analyzed using a flow cytometer (Troiano et al., 2007 (link)).

Cells were suspended in PBS containing Rhod2-AM (1 µM), incubated at 37°C for 15 min, and assessed by flow cytometry. In addition, cells were seeded in 4-well chambers and treated with Rhod2-AM at 37°C for 30 min. Images were captured on a confocal microscope using the Laser Scanning Microscope 5 PASCAL software (Carl Zeiss, Jena, Germany).

The mitochondrial membrane potential Δψ_m was determined by confocal image analysis and flow cytometry. SY-SH5Y cells were treated with 7,8-DHF at 5 μg/ml for 1 h. Then, 150 μM of glucose was added to the plate and the mixture was incubated for 24 h. After refreshing the media, JC-1 was added to each well and the cells were incubated for an additional 30 min at 37°C. After washing with phosphate-buffered saline (PBS), the stained cells were mounted onto microscope slides in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were captured on a confocal microscope using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany). For flow-cytometric measurement of Δψ_m, cells were harvested, washed, and suspended in PBS containing JC-1 (10 μg/ml). After incubation at 37°C for 15 min, the cells were washed, suspended in PBS, and analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA).