A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses
| Autor: | Ching-Ting Ma, Wan-Jou Shen, Chiou Feng Lin, Kao-Chi Yang, Chia-Yuan Hsieh, Chia Ling Chen, Pui-Ching Choi |
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| Rok vydání: | 2012 |
| Předmět: |
Biophysics
Oxidative phosphorylation Biology medicine.disease_cause Biochemistry Fluorescence Flow cytometry chemistry.chemical_compound Fixatives Dichlorofluorescein Cell Line Tumor medicine Humans Hydrogen peroxide Molecular Biology A549 cell chemistry.chemical_classification Reactive oxygen species medicine.diagnostic_test Staining and Labeling Methanol Proteins Cell Biology Flow Cytometry Fluoresceins Molecular biology Immunohistochemistry Oxidative Stress chemistry Reactive Oxygen Species Oxidation-Reduction Immunostaining Oxidative stress |
| Zdroj: | Biochemical and biophysical research communications. 430(1) |
| ISSN: | 1090-2104 |
| Popis: | 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 (H(2)O(2))-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), a chloromethyl derivative of H(2)DCFDA, 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 H(2)O(2)-, 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 H(2)O(2)-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. |
| Databáze: | OpenAIRE |
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