In this study, 4′,6-diamidino-2-phenylindole (DAPI)-stained cells were used to examine the changes in cell morphology during apoptosis under fluorescence microscopy. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline, then incubated with 30 min DAPI (1 μg/mL). In order to check the percentage of apoptotic nuclei, 200 to 300 cells were scored using a fluorescent microscope. Cell-cycle distribution of the cells was analyzed by propidium iodide staining and FACS analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific Inc., Waltham, MA, USA).
The fluorescent probes of 10 μM 2′, 7′-dichlorofluorescein diacetate (H2DCFDA, Molecular Probes) were used to measure hydrogen peroxide (H2O2), which represented the intracellular accumulation of ROS. The cells with H2DCFDA staining were subjected to a FACS analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific Inc., Waltham, MA USA). The data of the fluorescent intensity and the number of stained cells were quantified and analyzed and were represented as a percentage of the untreated control group in three independent experiments [24 (link)].
The fluorescent probes of 10 μM 2′, 7′-dichlorofluorescein diacetate (H2DCFDA, Molecular Probes) were used to measure hydrogen peroxide (H2O2), which represented the intracellular accumulation of ROS. The cells with H2DCFDA staining were subjected to a FACS analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific Inc., Waltham, MA USA). The data of the fluorescent intensity and the number of stained cells were quantified and analyzed and were represented as a percentage of the untreated control group in three independent experiments [24 (link)].
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