Real-time flow cytometry analysis of permeability transition in isolated mitochondria.
Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.
Lecoeur
Hervé
H
Theraptosis Research Laboratory, Theraptosis S.A., Institut Pasteur, 75015 Paris, France.
Langonné
Alain
A
Baux
Ludwig
L
Rebouillat
Dominique
D
Rustin
Pierre
P
Prévost
Marie-Christine
MC
Brenner
Catherine
C
Edelman
Léna
L
Jacotot
Etienne
E
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles; D002118:Calcium / Q000494:pharmacology; D002232:Carbocyanines; D002463:Cell Membrane Permeability; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure*; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007473:Ion Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D000408:Alamethicin; D002118:Calcium