10-N-Nonyl acridine orange (NAO) is an acridine orange derivative and is generally used as a fluorescent marker of the inner mitochondrial membrane in whole cells. It is believed to bind to negatively charged phospholipids. NAO accumulation in the cell seems to be related to specific interactions with mitochondrial membrane proteins and/or lipids, such as cardiolipin, and is largely independent of mitochondrial membrane potential.
Rhodamine 123 can be used in multi-parametric analysis, without fluorescence interference, in combination with common protein labeling dyes such as cyanine-5 and AMCA. With respect to apoptosis, the presence of mitochondrial membrane potential can be probed with rhodamine 123 while the structure and integrity of mitochondria can be assessed using 10-N-nonyl-acridine orange.
H. A. J. , R. 10-N nonyl-acridine orange: a fluorescent probe which stains mitochondria independently of their energetic state. Biochem. Biophys. Res. Commun.
Louis, MO, USA), and 2,7-dihydrodichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HEt), and dihydrorhodamine 123 (DHR123) were purchased from Molecular Probes (Eugene, OR, USA). A protease inhibitor cocktail was purchased from Roche (Boehringer Mannheim, Germany). Polyvinylidene difluoride (PVDF) membrane was purchased from Millipore (Bedford, MA, USA). Protein Assay Dye Reagent was obtained from Bio-Rad Laboratories (Hercules, CA, USA). The phenol red-free RPMI 1640 medium, penicillin/streptomycin, sodium pyruvate, calcein AM, Hank’s balanced salt solution (containing calcium) (HBSS/Ca), and 10-N-nonyl-acridine orange.
As the head group forms such compact bicycle structure, the head group area is quite small relative to the big tail region consisting of 4 acyl chains. Based on this special structure, the fluorescent mitochondrial indicator, nonyl acridine orange (NAO) was introduced in 1982, and was later found to target mitochondria by binding to CL. NAO has a very large head and small tail structure which can compensate with cardiolipin’s small head large tail structure, and arrange in a highly ordered way.
Adherent cells treated with H2O2 were rinsed twice with PBS and probed with fluorescent dyes (all from Molecular Probes) prepared in serum-free medium. To detect intracellular ROS, cells were stained with 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for 20 min at 370C. Cellular peroxide levels were assessed by staining with 30 μM dihydrorhodamine 123 (DHR 123) for 30 min at 370C. Mitochondrial membrane potential was measured after cell staining with 10 μM Rhodamine 123 (Rho123) for 30 min at 370C. Mitochondrial mass was determined after cell staining with 10 μM 10-n-Nonyl-Acridine Orange.
Mitochondrial abundance in C2C12 myotubes was assessed by 10-N-nonyl acridine orange (NAO) dye (Life Sciences, PA, USA) according to manufacturer’s instruction. After desired treatment, cells in 96-well plates were treated with 10