Apoptosis

, Volume 12, Issue 5, pp 803–813 | Cite as

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

  • Lorenzo Galluzzi
  • Naoufal Zamzami
  • Thibault de La Motte Rouge
  • Christophe Lemaire
  • Catherine Brenner
  • Guido Kroemer
Article

Abstract

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨm). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.

Keywords

Apoptosis FACS Fluorescence microscopy Fluorochromes Mitochondrial membrane permeabilization Mitochondrial transmembrane potential 

Abbreviations

1H-NMR

proton nuclear magnetic resonance

ΔΨm

mitochondrial transmembrane potential

AIF

apoptosis-inducing factor

ANT

adenine nucleotide translocase

APAF-1

apoptosis protease activating factor 1

BH3

Bcl-2 homology domain 3

CMXRos

chloromethyl-X-rosamine

CypD

cyclophilin D

Cyt c

cytochrome c

DiOC6(3)

3,3′dihexiloxalocarbocyanine iodide

ELISA

enzyme-linked immunosorbent assay

FACS

fluorescence-activated cell sorter

FSC

forward scatter

GFP

green fluorescent protein

HIV-1

human immunodeficiency virus type 1

HPLC

high-pressure liquid chromatography

HSP-60

heat shock protein of 60 kDa

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide

Omi/HtrA2

Omi stress-regulated endoprotease/High temperature requirement protein A 2

PT

permeability transition

PTPC

permeability transition pore complex

Rh 123

rhodamine 123

Smac/DIABLO

second mitochondria-derived activator of caspase/direct IAP binding protein with a low pI

SSC

side scatter

tBid

truncated Bid

TMRE

tetramethylrhodamine ethyl ester

TMRM

tetramethylrhodamine methyl ester

VDAC

voltage-dependent anion channel

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Copyright information

© Springer Science + Business Media, LLC 2007

Authors and Affiliations

  • Lorenzo Galluzzi
    • 1
    • 2
    • 3
  • Naoufal Zamzami
    • 1
    • 2
    • 3
  • Thibault de La Motte Rouge
    • 1
    • 2
    • 3
  • Christophe Lemaire
    • 4
  • Catherine Brenner
    • 4
  • Guido Kroemer
    • 1
    • 2
    • 3
    • 5
  1. 1.INSERM, U848VillejuifFrance
  2. 2.Institut Gustave RoussyVillejuifFrance
  3. 3.Faculté de Médecine-Université Paris-Sud XIVillejuifFrance
  4. 4.Université de Versailles/SQY-CNRS UMR 8159VersaillesFrance
  5. 5.INSERM, U848, Institut Gustave Roussy, PR138 rue Camille DesmoulinsVillejuifFrance

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