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Updated: Aug 26, 2026
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
Published on: September 7, 2012
Real-time flow cytometry analysis of permeability transition in isolated mitochondria
Hervé Lecoeur1, Alain Langonné, Ludwig Baux
1Theraptosis Research Laboratory, Theraptosis S.A., Institut Pasteur, 75015 Paris, France.
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 (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.
Insights
Flow cytometry methods were developed to screen compounds affecting mitochondrial membrane permeabilization (MMP) and the permeability transition pore (PTP). This approach allows simultaneous evaluation of mitochondrial size, structure, and membrane potential during MMP.
Area of Science:
- Cell Biology
- Biochemistry
- Biophysics
Background:
- Mitochondrial membrane permeabilization (MMP) is crucial in cell death pathways like apoptosis and necrosis.
- The opening of the permeability transition pore (PTP) is a critical, rate-limiting step controlling MMP.
- Existing methods for studying MMP lack the sensitivity and quantitative capabilities for high-throughput screening.
Purpose of the Study:
- To develop and validate a flow cytometry (FC)-based approach for screening and studying MMP inducers and blockers.
- To simultaneously assess mitochondrial size, structure, and inner membrane electrochemical potential (DeltaPsi(m)) during MMP.
- To provide a sensitive and quantitative method for evaluating compounds that modulate MMP.
Main Methods:
- Utilized flow cytometry (FC) for fixed-time and real-time analysis of isolated mitochondria.
- Co-evaluated mitochondrial size (forward scatter, FSC) and structure (side scatter, SSC) changes during MMP.
- Employed the DeltaPsi(m)-sensitive dye JC-1 for fluorescence-based detection of mitochondrial membrane potential variations.
Main Results:
- Demonstrated that calcium and alamethicin induce mitochondrial swelling (increased FSC, decreased SSC) detectable by FC.
- Showcased the ability of FC to detect DeltaPsi(m) changes using JC-1 in response to PTP openers and inner MMP inducers like mClCCP.
- Validated FC-based measurements against spectrophotometric data for mitochondrial swelling.
Conclusions:
- Developed a simple, highly sensitive, and quantitative FC-based assay for studying MMP.
- The presented methods are suitable for screening and characterizing compounds that modulate MMP and PTP activity.
- This approach offers a powerful tool for drug discovery and understanding cell death mechanisms.
