Staining of mitochondrial membranes with 10-nonyl acridine orange MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs

Background: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridi ne orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with ora nge emitting mitochondrial membrane potential (Delta Psi(m)) probes, we fou nd dear responses to Delta Psi(m), altering drugs for both probes. Methods: The three probes were titrated to determine whether saturation pla yed a role in the response to drugs. The effects of a variety of Delta Psi( m), altering drugs were tested for MFG and MTG at probe concentrations of 2 0 nM and 200 nM and for NAO at 0.1 mu M and 5 mu M, using rhodamine 123 at 0.1 mu M as a reference probe. Results: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione mon oxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), ca rbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitropheno l (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluor escence intensity of cells stained with 0.1 mu M NAO increased following in cubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin , and valinomycin. The results with 5 mu M NAO were similar. Conclusions: MFG, MTG, and NAO appeared poor choices for the membrane poten tial independent analysis of mitochondrial membrane mass. Considering the m olecular structure of these probes that factor accumulation in the mitochon drial membrane because of a positive charge, our results are not surprising . Published 2000 Wiley-Liss, Inc.