Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass Jake Jacobson,* , à Michael R. Duchenà and Simon J. R. Heales* ,   Departments of *Molecular Pathogenesis and  Clinical Biochemistry, Institute of Neurology, National Hospital for Neurology and Neurosurgery, London àDepartment of Physiology, University College London, UK Abstract 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 dam- age to the phospholipid may have consequences for mito- chondrial 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 mito- chondria 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 accu- mulation 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 dem- onstrate 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. Keywords: cardiolipin, fluorescence, membrane potential, mitochondria, nonyl acridine orange. J. Neurochem. (2002) 82, 224–233. The current renaissance of mitochondrial biology has established mitochondria as key organelles in determining cell fate. Not only do mitochondria provide eukaryotic cells with the bulk of the ATP necessary to sustain cellular function, it is now also clear that mitochondria may sense and transduce pro- and anti-apoptotic signals, thus assuming a role in both apoptotic and necrotic cell death. Additionally, mitochondria are tightly integrated into cellular calcium signalling and are the prime intracellular producers of reactive oxygen species. There is therefore great interest in the behaviour of mitochondria within living cells, and powerful techniques such confocal microscopy and fluores- cence activated cell sorting (FACS) have recently provided a vast amount of information on mitochondrial physiology. Fluorescence imaging and FACS make use of fluorescent probesthatlocalisetomitochondriaandcanbeusedtoassess mitochondrial membrane potential (DY m ), mitochondrial calcium uptake and mitochondrial movement and distribu- tionwithinlivingcells.Mostmitochondrialdyesmakeuseof the DY m in order to ÔtargetÕ the probes to mitochondria. In fact, no fluorescent dye specifically targets mitochondria. All are lipophilic cations, and due to a delocalised positive chargecompartmentalisewithincellsinresponsetogradients between the plasma membrane and the mitochondria where they concentrate according to Nernstian principles (Emaus et al. 1986; Loew et al. 1993; Duchen et al. 2002). The redistribution of these fluorophores upon mitochondrial Received January 23, 2002; revised manuscript received March 15, 2002; accepted March 18, 2002. Address correspondence and reprint requests to Jake Jacobson, Department of Molecular Pathogenesis, Institute of Neurology, National Hospital for Neurology and Neurosurgery, London. E-mail: j.jacobson@ucl.ac.uk Abbreviations used: CCD, cooled charge-coupled device; DMEM, Dulbecco’smodifiedEagle’smedium;FACS,fluorescenceactivatedcell sorting; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone; GFP, green fluorescent protein; NAO, nonyl acridine orange; TMRM, tetramethyl rhodamine methyl ester. Journal of Neurochemistry , 2002, 82, 224–233 224 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 82, 224–233