Intracellular Distribution of the Fluorescent Dye Nonyl Acridine Orange Responds to the Mitochondrial Membrane Potential: Implications for Assays of Cardiolipin and Mitochondrial Mass

J Neurochem. 2002 Jul;82(2):224-33. doi: 10.1046/j.1471-4159.2002.00945.x.


Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridine Orange / analogs & derivatives*
  • Acridine Orange / chemistry
  • Acridine Orange / metabolism*
  • Acridine Orange / pharmacokinetics
  • Animals
  • Animals, Newborn
  • Astrocytes / cytology
  • Astrocytes / drug effects
  • Astrocytes / metabolism
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / pharmacology
  • Cardiolipins / analysis*
  • Cardiolipins / metabolism
  • Cells, Cultured
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism*
  • Fluorescent Dyes / pharmacokinetics
  • Glutaral
  • Green Fluorescent Proteins
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Male
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology*
  • Mitochondria / chemistry
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Myocardium / cytology
  • Myocardium / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Rats, Wistar
  • Tissue Fixation
  • Transfection
  • Uncoupling Agents / pharmacology


  • Cardiolipins
  • Fluorescent Dyes
  • Luminescent Proteins
  • Uncoupling Agents
  • Green Fluorescent Proteins
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
  • N(10)-nonylacridine orange
  • Acridine Orange
  • Glutaral