A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

Wan-Jou Shen, Chia-Yuan Hsieh, Chia-Ling Chen, Kao-Chi Yang, Ching-Ting Ma, Pui-Ching Choi, Chiou Feng Lin

研究成果: 雜誌貢獻文章同行評審

11 引文 斯高帕斯(Scopus)


The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H2O2)-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a chloromethyl derivative of H2DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H2O2-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H2O2-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.
頁(從 - 到)442-447
期刊Biochemical and Biophysical Research Communications
出版狀態已發佈 - 1月 4 2013

ASJC Scopus subject areas

  • 生物化學
  • 生物物理學
  • 細胞生物學
  • 分子生物學


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