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

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)442-447
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume430
Issue number1
DOIs
Publication statusPublished - Jan 4 2013

Fingerprint

Reactive Oxygen Species
Staining and Labeling
Phosphorylation
Acetic Acid
Methanol
Assays
Fluorescence
Antimycin A
Oxidation
Fixatives
src Homology Domains
Flow cytometry
p38 Mitogen-Activated Protein Kinases
Starvation
Fluorescent Dyes
Hydrogen Peroxide
Flow Cytometry
Synchronization
Coloring Agents
Derivatives

Keywords

  • DCF
  • Fixative
  • Flow cytometry
  • Immunostaining
  • MAPK
  • ROS

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses. / Shen, Wan-Jou; Hsieh, Chia-Yuan; Chen, Chia-Ling; Yang, Kao-Chi; Ma, Ching-Ting; Choi, Pui-Ching; Lin, Chiou Feng.

In: Biochemical and Biophysical Research Communications, Vol. 430, No. 1, 04.01.2013, p. 442-447.

Research output: Contribution to journalArticle

@article{d1f8234496cc40068b5ca0d8f9fd8753,
title = "A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses",
abstract = "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.",
keywords = "DCF, Fixative, Flow cytometry, Immunostaining, MAPK, ROS",
author = "Wan-Jou Shen and Chia-Yuan Hsieh and Chia-Ling Chen and Kao-Chi Yang and Ching-Ting Ma and Pui-Ching Choi and Lin, {Chiou Feng}",
year = "2013",
month = "1",
day = "4",
doi = "10.1016/j.bbrc.2012.11.037",
language = "English",
volume = "430",
pages = "442--447",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Elsevier B.V.",
number = "1",

}

TY - JOUR

T1 - A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

AU - Shen, Wan-Jou

AU - Hsieh, Chia-Yuan

AU - Chen, Chia-Ling

AU - Yang, Kao-Chi

AU - Ma, Ching-Ting

AU - Choi, Pui-Ching

AU - Lin, Chiou Feng

PY - 2013/1/4

Y1 - 2013/1/4

N2 - 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.

AB - 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.

KW - DCF

KW - Fixative

KW - Flow cytometry

KW - Immunostaining

KW - MAPK

KW - ROS

UR - http://www.scopus.com/inward/record.url?scp=84872377312&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84872377312&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2012.11.037

DO - 10.1016/j.bbrc.2012.11.037

M3 - Article

VL - 430

SP - 442

EP - 447

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -