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High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate
Alice Wong1, Gino A Cortopassi
1Department of Molecular Biosciences, University of California, Davis 95616, USA.
Abstract:
Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP, resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.
Insights
We developed a new high-throughput method to measure mitochondrial membrane potential (MMP) in neural cells. This assay can help identify drugs that enhance mitochondrial function and screen for genetic mutations affecting MMP.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Mitochondrial genetic diseases are linked to decreased mitochondrial membrane potential (MMP).
- Accurate measurement of MMP is crucial for understanding mitochondrial function and disease.
- Existing methods for MMP assessment are often low-throughput and time-consuming.
Purpose of the Study:
- To develop a high-throughput assay for measuring MMP in intact neural cells.
- To validate the assay using known MMP-modulating agents and a mtDNA-deficient cell line.
- To enable drug screening for compounds that modulate MMP and genetic screening for MMP-related mutations.
Main Methods:
- Utilized tetramethylrhodamine, methyl ester (TMRM), a potentiometric dye, in a 48-well plate format for high-throughput MMP measurement.
- Applied known MMP depolarizing agents (FCCP, DNP) and non-mitochondrial affecting drugs to validate assay specificity.
- Assessed MMP in a cell line with mitochondrial DNA (mtDNA) deficiency.
Main Results:
- The TMRM-based assay demonstrated a time- and concentration-dependent decrease in fluorescence upon addition of depolarizing agents, with saturable kinetics.
- Drugs affecting non-mitochondrial properties did not alter fluorescence, confirming assay specificity.
- The mtDNA-deficient cell line exhibited reduced fluorescence, which was not further decreased by depolarizing agents, consistent with impaired mitochondrial function.
Conclusions:
- The developed 48-well plate assay provides a robust, high-throughput method for measuring MMP in neural cells.
- This system facilitates the discovery of small molecules that enhance mitochondrial function.
- The assay is suitable for screening neural cell populations for genetic mutations impacting MMP.