Ix70 fv500 confocal microscope

The FDA/PI dye combination discriminates between living and dead cells. Living cells convert FDA to fluorescent fluorescein and exhibit green fluorescence, while dead cells are stained by a membrane impermeable PI and exhibit red fluorescence. This staining and visualization protocol ensured the elimination of the previously described limitations [35 (link)]. HT-29 cells were stained with freshly prepared solutions of 0.125 μg/ml FDA and 0.5 μg/ml PI for 15 minutes (Sigma-Aldrich Corp., St. Louis, MO, USA). Then, the samples were analysed using an Olympus IX70 FV500 confocal microscope with a 10x UPlanApo lens. Fluorescence was recorded in the sequential mode to eliminate potential fluorescence bleed-through. The FDA and PI fluorescence excited by an Ar laser with a wavelength of 488 nm and a He-Ne laser with a wavelength of 543 nm, respectively, was collected by 505–525 nm (for FDA) and 560–610 nm (for PI) BP filters [35 (link)].
Quantitative data analysis was performed using Fluoview500 software (version 5.0), Olympus, Shinjuk, Tokyo, Japan) and ImageJ (Sun Microsystems, Santa Clara, California) as described previously [36 ].

Mitochondria were visualized with MitoTracker DeepRed dye (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which stains mitochondria in living cells regardless of mitochondrial membrane potential. The dye solution was added to wells, and plates were incubated for 10 minutes at 37°C. The confocal images were collected with the Olympus IX70 FV500 confocal microscope equipped with an oil 60x UPlanApo lens. A He-Ne 633 nm laser used as a light source, and the fluorescence signal was collected through the 660 nm BA filter.

Dihydrorhodamine 123 (DHR123) was used to assess the ROS level. After reacting with ROS, this dye is oxidized to fluorescent rhodamine 123 (R123). A solution of 12.5 μM DHR123 in PBS was added to each well. The cells were incubated for 15 min at 37°C. The hydrogen peroxide (H₂O₂) was used as a positive control (15 μM). Cell fluorescence was observed using an argon laser with a wavelength of 488 nm and a 505–525 nm BP filter. Observations were made with an Olympus IX70 FV500 confocal microscope with a 40x UPlanApo lens within one hour. After that time, the fluorescence intensity decreases compared with the initial value. Because DHR123 is a cationic dye and accumulates in mitochondria, it is sensitive to changes in mitochondrial mass. As was suggested by Forkink et al., the mitochondrial membrane potential (∆Ψm) should be assessed in parallel [37 (link)].