Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein

Methods Mol Biol. 2021:2274:207-215. doi: 10.1007/978-1-0716-1258-3_18.

Abstract

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.

Keywords: Fluorescence microscope; Hydroxyphenyl fluorescein; Image analysis; Oncogenic RAS; ROS; Transform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism*
  • Fluoresceins / chemistry*
  • Fluorescence*
  • Fluorescent Dyes / chemistry*
  • Hydroxyl Radical / analysis
  • Mice
  • Mice, Knockout
  • NIH 3T3 Cells
  • Oxidation-Reduction
  • Oxidative Stress
  • Peroxynitrous Acid / analysis
  • Reactive Oxygen Species / metabolism*
  • Spectrometry, Fluorescence
  • Tumor Suppressor Protein p53 / physiology*

Substances

  • Fluoresceins
  • Fluorescent Dyes
  • Reactive Oxygen Species
  • Trp53 protein, mouse
  • Tumor Suppressor Protein p53
  • hydroxyphenyl fluorescein
  • Peroxynitrous Acid
  • Hydroxyl Radical