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Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro
Published on: July 17, 2018
Fluorometric methods for detection of mitochondrial membrane permeabilization in apoptosis
Soumya Sinha Roy1, György Hajnóczky
1Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|July 18, 2009
Summary
This study details fluorescence measurements to assess mitochondrial membrane integrity and cell death pathways. These methods quantify the release of factors like cytochrome c, crucial for understanding apoptosis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Mitochondrial regulation of cell death involves the release of proapoptotic factors via outer mitochondrial membrane (OMM) disruption.
- The Bcl-2 protein family controls OMM permeability, influencing apoptosis.
- Inner mitochondrial membrane (IMM) permeability transition also impacts OMM integrity.
Purpose of the Study:
- To provide a manual for fluorescence measurements evaluating mitochondrial membrane barrier function.
- To assess the release of proapoptotic factors and downstream caspase activation.
- To differentiate mechanisms of OMM permeabilization.
Main Methods:
- Real-time, quantitative fluorescence measurements in permeabilized cells or isolated mitochondria.
- Measurement of mitochondrial membrane potential, Ca(2+) and cytochrome c retention, and matrix volume.
- Protocols for evaluating caspase activation and discriminating OMM permeabilization mechanisms.
Main Results:
- Established fluorescence-based assays for OMM and IMM barrier function.
- Quantified release of key proapoptotic factors like cytochrome c.
- Demonstrated methods to distinguish between different OMM permeabilization pathways.
Conclusions:
- Fluorescence measurements are effective tools for studying mitochondrial cell death pathways.
- Understanding OMM and IMM dynamics is critical for apoptosis research.
- The provided protocols facilitate detailed analysis of mitochondrial outer membrane permeabilization.
