Fv3000 ix81 confocal microscope

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).

In cells, reactive oxygen species (ROS) were determined using a fluorescent dye protocol [59 (link)]. Cells were treated with different concentrations of each extract for 1 h and then incubated with H₂O₂ (100 µM) for 1 h. The DCF fluorescence intensity was detected on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation at 488 nm and emission at 535 nm. The confocal imaging was 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).