ROS production was evaluated using 2′,7′-dichlorofluorescein diacetate (DCFDA), as described previously40 (link). The oxidation of the dye by ROS generates a highly fluorescent compound, 2,7-dichlorofluorescein (DCF) that can be detected at λex = 504 nm and λem = 529 nm. Briefly, cells were incubated with DCFDA (5 µM) for 30 minutes, washed with warm PBS/5% FBS, and then imaged in a 37 °C, CO2 atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare, Little Chalfont, UK). The acquired images were then analyzed with Developer Toolbox 1.9.2 (GE Healthcare).
Lipid peroxidation was measured with BODIPY®, following the manufacturer’s instructions (Thermo Fisher Scientific). Upon oxidation by lipid hydroperoxides, BODIPY® changes its maximum fluorescence emission wavelength from 590 nm to 510 nm. Briefly, cells were incubated with BODIPY® (10 µM) for 30 minutes, washed with PBS, and then visualized in a 37 °C, CO2 atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare). The acquired images were analyzed with CellProfilerTM.
Lipid peroxidation was measured with BODIPY®, following the manufacturer’s instructions (Thermo Fisher Scientific). Upon oxidation by lipid hydroperoxides, BODIPY® changes its maximum fluorescence emission wavelength from 590 nm to 510 nm. Briefly, cells were incubated with BODIPY® (10 µM) for 30 minutes, washed with PBS, and then visualized in a 37 °C, CO2 atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare). The acquired images were analyzed with CellProfilerTM.
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