Inverted lsm 780 laser scanning confocal microscope

Cells were seeded at a density of 6× 10⁴ cells per well in glass bottom cell culture dishes and incubated with different treatments for 48 hours. After incubation, the culture medium was discarded, and the cells were washed with RPMI 1640 3 times. Then, RPMI 1640 containing 1 µM FerroOrange (Dojindo, Japan, Cat. F374) was added and the cells were incubated for 30 minutes at 37°C in the dark. Finally, the Fe²⁺ content in the cells was analysed using a fluorescence microplate reader (ex 543 nm, em 580 nm) (Tecan Trading AG, Switzerland). In addition, focal images were acquired with a Zeiss inverted LSM 780 laser scanning confocal microscope (Zeiss) using a 100x/1.4 DIC Plan-Apochromat oil immersion objective. Three representative fields were captured for each condition using identical exposure times. Images were obtained with a Cy3 filter (excitation and emission wavelengths 514 and 525-596 nm, respectively). The resolution of the obtained images is 1024×1024 pixels, with a pixel depth of 8 bits, a pinhole size of 1 Airy unit, and a line averaging of 2.

To measure lipid peroxidation in cells, we used a peroxidation malondialdehyde (MDA) assay kit (Cat. No. S0131M, Beyotime Biotech, China) according to the manufacturer's protocol. In brief, after 48 hours of incubation with various treatments, cells were harvested by centrifugation at 1,000 rpm at 4°C for 5 minutes. The supernatant was collected, and thiobarbituric acid (TBA) reagent was added at a ratio of 1:2, after which the mixture was heated at 100°C for 15 minutes. After heating, the mixture was cooled to room temperature and centrifuged at 1000 g at room temperature for 10 minutes. After centrifugation, the supernatant was removed, and the absorbance was measured at 532 nm using an enzyme-labelled instrument. At the same time, intracellular lipid hydroperoxide levels were measured using a selective Liperfluo probe whose concentration was set at 10 μM (Ex = 532 nm, Em = 535-650 nm) and a Zeiss inverted LSM 780 laser scanning confocal microscope (Zeiss) using a 100x/1.4 DIC Plan-Apochromat oil immersion objective. Three representative fields were captured using identical exposure times for each experimental condition. Images were obtained with the FITC filter (ex 488 nm, em 525 nm). The images were obtained at a resolution of are 1024×1024 pixels with a pixel depth of 8 bits, a pinhole size of 1 Airy unit, and a line averaging of 2.