Open AccessStaining of mitochondrial membranes with 10‐nonyl acridine orange MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs
Author(s) -
Keij Jan F.,
BellPrince Carolyn,
Steinkamp John A.
Publication year - 2000
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(20000301)39:3<203::aid-cyto5>3.0.co;2-z
Subject(s) - nigericin , acridine orange , protonophore , gramicidin , valinomycin , membrane potential , antimycin a , chemistry , oligomycin , membrane , biophysics , biochemistry , rhodamine 123 , inner mitochondrial membrane , mitochondrion , biology , atpase , apoptosis , multiple drug resistance , enzyme , antibiotics
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 (ΔΨ m ) probes, we found clear responses to ΔΨ 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 ΔΨ m altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 μM and 5 μM, using rhodamine 123 at 0.1 μM 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 μM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 μM 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.