Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2',7'-dichlorofluorescin diacetate (DCFDA) assay

Daniela Figueroa, Mohammad Asaduzzaman, Fiona Young

Research output: Contribution to journalArticle

12 Citations (Scopus)
10 Downloads (Pure)

Abstract

The detection of reactive oxygen species (ROS) using 2′,7′–dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 μM) for 45 min, and then exposed to TBHP (0-50 μM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 μM DCFDA with 25 μM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology.

Original languageEnglish
Pages (from-to)26-33
Number of pages8
JournalJOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS
Volume94
DOIs
Publication statusPublished - 2018

Bibliographical note

Author's post-print must be released with a Creative Commons Attribution Non-Commercial No Derivatives License

Keywords

  • Reactive oxygen species (ROS)
  • Breast cancer
  • cell viability

Fingerprint Dive into the research topics of 'Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2',7'-dichlorofluorescin diacetate (DCFDA) assay'. Together they form a unique fingerprint.

Cite this