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

MitoTracker dyes are fluorescent mitochondrial markers that covalently bind free sulfhydryls. The impact of alterations in mitochondrial membrane potential (Delta Psi(m)) 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. H(2)O(2) pretreatment led to a consistent increase in neuronal MitoTracker Orange and Red and astrocytic MitoTracker Green and Orange fluorescence intensity. H(2)O(2) 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 Delta Psi(m) 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 Delta Psi(m) and oxidation negates their use as markers of mitochondrial dynamics in healthy cultures.