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Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader
J R Blattner1, L He, J J Lemasters
1Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA.
Analytical Biochemistry
|August 8, 2001
Summary
We developed a high-throughput assay to screen compounds affecting mitochondrial permeability transition (MPT). This method efficiently measures mitochondrial swelling, membrane potential, and calcium release, aiding in MPT research.
Area of Science:
- Mitochondrial Physiology
- Biochemistry
- Pharmacology
Background:
- The opening of mitochondrial permeability transition (PT) pores leads to mitochondrial permeability transition (MPT), characterized by swelling, membrane depolarization, and solute release.
- Understanding MPT is crucial for cellular health and disease, necessitating efficient screening methods for modulators.
Purpose of the Study:
- To develop high-throughput assays using a fluorescence plate reader for screening potential inducers and blockers of the mitochondrial permeability transition (MPT).
- To establish a method capable of simultaneously measuring multiple MPT-related events, including calcium uptake, membrane potential, and mitochondrial swelling.
Main Methods:
- Isolated rat liver mitochondria were incubated in multiwell plates with fluorescent indicators for mitochondrial membrane potential (TMRM) and Ca(2+) (Fluo-5N).
- Mitochondrial swelling was monitored by light absorbance changes, and Ca(2+) release was measured via fluorescence.
- Carboxydichlorofluorescein (carboxy-DCF) was used to monitor solute release, allowing differentiation of MPT modulators from other mitochondrial inhibitors.
Main Results:
- The assay successfully monitored mitochondrial polarization, Ca(2+) uptake, depolarization, Ca(2+) release, and swelling in response to stimuli.
- Cyclosporin A confirmed the MPT pathway, as it prevented swelling, depolarization, and Ca(2+) release.
- Measurements of Ca(2+) uptake, membrane potential, and swelling were independent and could be performed rapidly in a 48-well plate format.
Conclusions:
- A robust, high-throughput multiwell assay was developed for screening compounds that modulate the mitochondrial permeability transition (MPT).
- This assay enables simultaneous measurement of key MPT events, facilitating the identification of novel MPT inducers and blockers.
- The method allows for clear distinction between MPT modulators and other mitochondrial affecting agents.