MitoSOX Red is a
fluorescent probe used for the detection of mitochondrial
reactive oxygen species by live cell imaging. The lipophilic, positively charged
triphenylphosphonium moiety within
MitoSOX concentrates the
superoxide-sensitive
dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common
MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines.
MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5-10μM
MitoSOX caused severe loss of
ATP synthesis-linked respiration. Redistribution of
MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling.
MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with
MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar
MitoSOX had no adverse effects. The
MitoSOX parent compound
dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating
dihydroethidium with
cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of
MitoSOX or
dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because
MitoSOX is inherently capable of causing severe
mitochondrial dysfunction with the potential to alter
superoxide production, its use therefore requires careful optimization in imaging protocols.