Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition

Anal Biochem. 2017 Mar 1:520:44-48. doi: 10.1016/j.ab.2017.01.001. Epub 2017 Jan 4.

Abstract

Peroxynitrite (ONOO-), a strong oxidant species, is produced by the reaction of nitric oxide (NO) and superoxide (O2.-) radicals. It plays an important role as a biological regulator in numbers of physiological and pathological processes. In this study, we developed fluorescence-based real-time quantitative measurements to detect intracellular ONOO-. The probe DAX-J2 PON Green showed high selectivity toward ONOO- over other competing species, and has been successfully applied in microplate reader and flow cytometer to quantitatively measure endogenous ONOO- production. Moreover, the results demonstrated the inhibitory effects of curcumin on intracellular ONOO- generation.

Keywords: Curcumin; Flow cytometer; Fluorescence; Microplate reader; Peroxynitrite.

MeSH terms

  • Animals
  • Flow Cytometry
  • Fluorescent Dyes / chemistry
  • HeLa Cells
  • Humans
  • Jurkat Cells
  • Mice
  • Microscopy, Fluorescence
  • Nitric Oxide / chemistry
  • Peroxynitrous Acid / analysis*
  • Peroxynitrous Acid / metabolism
  • RAW 264.7 Cells
  • Superoxides / chemistry
  • Tissue Array Analysis*

Substances

  • Fluorescent Dyes
  • Superoxides
  • Peroxynitrous Acid
  • Nitric Oxide