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

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.