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

Cytometry. 2000 Mar 1;39(3):203-10. doi: 10.1002/(sici)1097-0320(20000301)39:3<203::aid-cyto5>;2-z.


Background: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes.

Methods: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM as a reference probe.

Results: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM NAO were similar.

Conclusions: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000. Published 2000 Wiley-Liss, Inc.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 2,4-Dinitrophenol / pharmacology
  • Acridine Orange
  • Aldehydes
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / pharmacology
  • Cell Line
  • Diacetyl / analogs & derivatives
  • Diacetyl / pharmacology
  • Flow Cytometry / methods
  • Fluorescent Dyes*
  • HL-60 Cells
  • Humans
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / physiology*
  • Intracellular Membranes / ultrastructure
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Mitochondria / drug effects
  • Mitochondria / physiology*
  • Mitochondria / ultrastructure
  • Nigericin / pharmacology
  • Pyridinium Compounds
  • U937 Cells
  • Uncoupling Agents / pharmacology*


  • Aldehydes
  • Fluorescent Dyes
  • MitoFlour green
  • Pyridinium Compounds
  • Uncoupling Agents
  • mitotracker green FM
  • diacetylmonoxime
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Acridine Orange
  • Diacetyl
  • 2,4-Dinitrophenol
  • Nigericin