Frontiers of Innovative Research in Science and Technology, Konan University, Kobe, Japan.
Methods Mol Biol. 2021;2274:207-215. doi: 10.1007/978-1-0716-1258-3_18.
Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.
已经开发出各种用于检测细胞内活性氧（ROS）的荧光探针，因为 ROS 水平与细胞状态密切相关。在这里，我们描述了一种使用荧光探针羟苯基荧光素（HPF）检测活细胞内 ROS 的方法，HPF 可以检测羟基自由基和过氧亚硝酸盐。通过逆转录病毒系统表达致癌 RAS，将 NIH3T3 细胞和 p53 缺失（p53）小鼠胚胎成纤维细胞（MEF）转化。将细胞在 37°C 下用 HPF 处理 30 分钟，然后用 PBS 洗涤后，在 488nm 的激发波长下使用共聚焦荧光显微镜获取图像。
