Detection of reactive oxygen species during the cell cycle under normal culture conditions using a modified fixed-sample staining method

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

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

Original languageEnglish
Pages (from-to)149-161
Number of pages13
JournalJournal of Immunoassay and Immunochemistry
Volume36
Issue number2
DOIs
Publication statusPublished - Mar 4 2015

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Keywords

  • cell cycle
  • DCF
  • fixative
  • flow cytometry
  • immunostaining
  • ROS

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Clinical Biochemistry
  • Medical Laboratory Technology

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