Concomitant real-time monitoring of intracellular reactive oxygen species and mitochondrial membrane potential in individual living promonocytic cells

Naomi Zurgil, Yana Shafran, Elena Afrimzon, Dror Fixler, Asher Shainberg, Mordechai Deutsch

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Reactive oxygen species (ROS) have recently been shown to be involved in multiple physiological responses through modulation of signaling pathways. Inappropriate production of these radicals, and their metabolites, leads to the development of various pathologies. Free radicals can induce both positive and negative effects in cells, and their metabolic pathways are very complex. Hence, it is crucial to be able to simultaneously and directly determine their production dynamics and concentrations in individual living cells, in physiological or pathological states, and in response to drugs. The aim of the present study was to monitor in real time the rates of ROS generation in promonocytic cells upon stimulation with hydrogen peroxide and oxidized lipid. Quantitative detection of intracellular ROS concentration in intact living U937 cells was performed by fluorescence intensity (FI) and polarization (FP) measurements utilizing the Optical LiveCell™ Array technology. The "dihydro" derivative probes of fluorescein (DCF-DA) and rhodamine (DHR123) were used to assess the intracellular levels of ROS. Each probe molecule exhibited a characteristic FI and FP in its non-fluorescent or oxidized form. Analysis of the temporal relationship between the kinetics of ROS generation and the onset of changes in mitochondrial membrane potential shows high variability within a cell population with regard to both processes. The data demonstrated that temporal measurement of ROS generation, in identifiable individual cells, reveals kinetic behavior that otherwise would be undetected.

Original languageEnglish
Pages (from-to)27-41
Number of pages15
JournalJournal of Immunological Methods
Volume316
Issue number1-2
DOIs
StatePublished - 20 Oct 2006

Bibliographical note

Funding Information:
This research was supported by the Horowitz Foundation.

Keywords

  • 2,7-dichlorofluorescein diacetate
  • CCCP
  • DCF-DA
  • DHE
  • DHR123
  • FI
  • FLT
  • FP
  • IBC
  • ICS
  • Individual blood cells
  • L-NAME
  • LDL
  • LPC
  • LPS
  • Live Cell Array (LCA)
  • MMP
  • Mitochondrial membrane potential (MMP)
  • N-nitro-larginine methyl ester
  • OLCA
  • Optical LiveCell™
  • PI
  • RNS
  • ROS
  • Reactive oxygen species
  • Reactive oxygen species (ROS)
  • SOD
  • TMRM
  • carbonyl cyanide m-chloro-phenyl hydrazone
  • dihydroethidium
  • dihydrorhodamine123
  • fluorescence intensity
  • fluorescence lifetime
  • fluorescence polarization
  • individual blood cells
  • individual cell scanner
  • lipopolysaccharide
  • low-density lipoproteins
  • lysophosphatidylcholine
  • mitochondrial membrane potential
  • propidium iodide
  • reactive nitrogen species
  • superoxide dismutase
  • tetramethylrhodamine methyl ester

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