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Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Authors
Journal
Experimental Cell Research
0014-4827
Publisher
Elsevier
Publication Date
Volume
294
Issue
1
Identifiers
DOI: 10.1016/j.yexcr.2003.10.030
Keywords
  • Flow Cytometry
  • Mitochondria
  • Ptp
Disciplines
  • Chemistry
  • Philosophy

Abstract

Abstract 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 (ΔΨ 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 ΔΨ m-sensitive cationic lipophilic dye JC-1 permits to detect ΔΨ 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.

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