Fv10 asw 4.2 viewer software

ROS was measured by a reactive oxygen assay kit using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the fluorescence probe. For ROS imaging in cells, cells (A549 and A549T) were seeded at desired concentration in covered glass-bottomed confocal dishes (NEST Biotechnology Co. Ltd.), after cultured for at least 24 h, cells were washed once and incubated with spermidine (Spd, 50 μM) and spermine (Spm, 50 μM) in RPMI1640 containing 10%FBS for 6 hours, then cells were washed and incubated with DCFH-DA (10 μM) in RPMI1640 containing 10%FBS for 30 min. After washing with PBS, confocal imaging was performed on an OLYMPUS FV1000-IX81 confocal microscope (Olympus Corporation, Japan). Confocal images were processed by Olympus FV10-ASW 4.2 viewer software.

To detect the cellular localization of RinA, the DNA fragment of the full-length rinA ORF was amplified using PCR, and RinA was fused with eGFP at its C-terminal. The fusion gene was controlled by S. aureus S10 promoter. The RinA::eGFP fluorescent expression strain was confirmed by PCR and sequencing. An inverted confocal laser scanning microscope (FV1000, Olympus) was utilized for observation. An argon ion laser (Ex = 488 nm, Em = 515–530 nm) was used to observe fluorescent signals of eGFP. Finally, the confocal images were captured using the FV10-ASW 4.2 Viewer software (Olympus).

To detect the cellular localization of LcpB, we fused LcpB with GFP at its C-terminal tail with its promoter using pALC, and FM4-64 was used to determine the location of cell membrane. For confocal microscopy, an inverted confocal laser scanning microscope (FV1000, Olympus) was used. For the observation of fluorescent signals of GFP, an argon ion laser (Ex = 488 nm, Em = 515–530 nm) was used. For the observation of FM4-64, fluorescent signals were acquired using a He-Ne laser (Ex = 559 nm, Em = 570–670 nm). Finally, all the confocal images were captured with FV10-ASW 4.2 Viewer software (Olympus).

Mitochondria were visualized using CellLight™ Mitochondria-GFP, BacMam 2.0 (ThermoFisher Scientific, Waltham, MA, USA). To visualize mitochondrial movement in living cells, the HL-1 cells were cultured in NuncTM Glass Base Dish, 12 mm (ThermoFisher Scientific, Waltham, MA, USA). The HL-1 cells were incubated with a studied factor for 1 h (5 µM iFBP2, 10 µM GSK3 inhibitor SB216763, 1 µM PI3K inhibitor wortmannin, 5.5 mm glucose; Merck, Darmstadt, Germany) and images were acquired using the Time Scan option the FV-1000 confocal microscope. Time-lapse videos were created on 3× digital zoom and analyzed using FV-10-ASW 4.2 Viewer software (Olympus). To avoid the measurement of random movement of mitochondria powered by cytoplasm fluctuations only the organelles that moved at least 2 μm during the image acquisition time were taken into account [22 (link)]. If a mitochondrion moved with pausing episodes, the mean velocity was counted rejecting pause time. The experiments were replicated at least three times, and velocities of at least 482 mitochondria were measured for each treatment.
The length and shape of mitochondria were analyzed using the MiNA (Mitochondrial Network Analysis by Sturat Lab) plugin of the ImageJ/FIJI. At least 5480 mitochondria were measured in each condition.

RAW264.7 cells were plated in confocal dishes (35 mm × 12 mm, Φ 20 mm glass bottom) at a density of 2 × 10⁵ cells per dishes for 12 h in a 37 °C humidified incubator with 5% CO₂. Then, the cells were incubated with 1 mL of fresh medium containing LBP < 10 kDa or LBP > 10 kDa (800 μg/mL) for 24 h. After removing the sample medium, cells were incubated with 2.5 µg/mL CytoFlamma Fluor WGA (a fluorescent probe for labeling cell membrane, excitation at 488 nm), 100 nM Mito-Tracker Green (a fluorescent dye for staining mitochondria, excitation at 488 nm) or 100 nM DAPI (a fluorescent dye for staining nucleus, excitation at 405 nm) at 37 °C for another 30 min respectively. Then, the stained cells were washed with cold PBS three times and observed by confocal imaging performed on an OLYMPUS FV3000-IX81 confocal microscope (Olympus Corporation, Tokyo, Japan). Confocal images were processed by Olympus FV10-ASW 4.2 viewer software (Olympus Corporation, Tokyo, Japan). The average fluorescence intensity of mitochondria in cells and the average size of cell nucleus were quantitatively analyzed by ImageJ software (V1.8.0).