MitoTracker labeling in primary neuronal and astrocytic cultures: Influence of mitochondrial membrane potential and oxidants

Jennifer F. Buckman, Hélène Hernández, Geraldine J. Kress, Tatyana V. Votyakova, Sumon Pal, Ian J. Reynolds

Research output: Contribution to journalArticlepeer-review

153 Scopus citations

Abstract

MitoTracker dyes are fluorescent mitochondrial markers that covalently bind free sulfhydryls. The impact of alterations in mitochondrial membrane potential (ΔΨm) and oxidant stress on MitoTracker staining in mitochondria in cultured neurons and astrocytes has been investigated. p-(Trifluoromethoxy) phenyl-hydrazone (FCCP) significantly decreased MitoTracker loading, except with MitoTracker Green in neurons and MitoTracker Red in astrocytes. Treatment with FCCP after loading increased fluorescence intensity and caused a relocalization of the dyes. The magnitude of these effects was contingent on which MitoTracker, cell type and dye concentration were used. H2O2 pretreatment led to a consistent increase in neuronal MitoTracker Orange and Red and astrocytic MitoTracker Green and Orange fluorescence intensity. H2O2 exposure following loading increased MitoTracker Red fluorescence in astrocytes. In rat brain mitochondria, high concentrations of MitoTracker dyes uncoupled respiration in state 4 and inhibited maximal respiration. Thus, loading and mitochondrial localization of the MitoTracker dyes can be influenced by loss of ΔΨm and increased oxidant burden. These dyes can also directly inhibit respiration. Care must be taken in interpreting data collected using MitoTrackers dyes as these dyes have several potential limitations. Although MitoTrackers may have some value in identifying the location of mitochondria within cultured neurons and astrocytes, their sensitivity to ΔΨm and oxidation negates their use as markers of mitochondrial dynamics in healthy cultures.

Original languageEnglish
Pages (from-to)165-176
Number of pages12
JournalJournal of Neuroscience Methods
Volume104
Issue number2
DOIs
StatePublished - Jan 15 2001

Keywords

  • Fluorescence imaging
  • Mitochondria
  • Oxidative stress

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