Fv3000 laser scanning confocal microscope

A549 cells in the logarithmic growth phase were transferred to glass bottom dishes with a density in each dish of 1 × 10⁵. The cells grew in the cell culture incubator for 24 h. Then, we added the sample solution of compounds 1–6 with a concentration of 10 μM to each dish, respectively. After 30 min of dark incubation, the solution in the dishes was carefully sucked out with pipetting gun and washed with PBS buffer for three times. Finally, we added 1 mL PBS buffer to each dish and observed them under a 60-fold oil immersion lens using an FV3000 laser scanning confocal microscope (Zeiss). The procedure of mitochondrial colocalization imaging was similar to the above procedure. After the A549 cells were transferred to glass bottom dishes and cultivated in cell incubators for 24 h, the mixed solutions of compounds 1–6 (10 µM) and Mito Tracker Red (MT) (200 nM) were added, respectively. After 30 min of dark incubation in the incubator, the solution was sucked out carefully, and the dishes were washed three times by PBS buffer. Finally, 1 mL PBS buffer was added to each dish, and they were observed by an FV3000 laser scanning confocal microscope (Zeiss) under a 60-fold oil immersion lens.

A549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), then seeded in a glass bottom dish with a density of 1 × 10⁵ cells per dish and incubated in an incubator with 5% CO₂ for 24 h. Flavone derivatives (1000 μL, 10 μM) were added to the dish and incubated at 37 °C for 30 min, the medium was later removed, and the cells were washed three times with phosphate buffered saline (PBS) buffer. Then the cell imaging of these compounds were observed on a FV3000 laser scanning confocal microscope (Zeiss) using an oil immersion lens (60× magnification, NA 1.4) within a bandwidth of 460−560 nm. A549 cells were cultured for 24 h with the same way the above mentioned then flavone derivatives (10 μM) and 200 nM Mito Tracker Deep Red (a commercially available mitochondrial dye) were added to the dish and incubated for 30 min for colocalization experiments. Thirty min later, the solutions were removed and cells were washed with PBS three times. Last, cell imaging of these flavone derivatives were obtained on a confocal laser scanning microscope.