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Mitochondrial Morphofunctional Profiling in Primary Human Skin Fibroblasts Using TMRM and Mitotracker Green
Jesper M M Bergmans1, Els M A van de Westerlo2, Sander Grefte3
1Department of Pediatrics, Amalia Children's Hospital, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|November 15, 2024
Summary
This study introduces a method using TMRM and Mitotracker Green FM dyes to analyze mitochondrial function, specifically morphology and membrane potential (Δψ), in living cells. The protocol is optimized for primary human skin fibroblasts using epifluorescence microscopy.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Biophysics
Background:
- Mitochondrial morphology and membrane potential (Δψ) are crucial indicators of cellular health and function.
- Assessing these parameters in living cells requires reliable and integrated analytical methods.
- Fluorescent lipophilic cations offer a means to visualize and quantify mitochondrial status based on Δψ.
Purpose of the Study:
- To present an integrated protocol for the simultaneous analysis of mitochondrial morphology and semiquantitative membrane potential (Δψ) in living cells.
- To optimize the use of tetramethylrhodamine methyl ester (TMRM) and Mitotracker Green FM (MG) for this dual analysis.
- To demonstrate the efficacy of this approach in primary human skin fibroblasts (PHSFs).
Main Methods:
- Utilizing epifluorescence microscopy for imaging.
- Co-staining of PHSFs with TMRM (for Δψ) and MG (for morphology).
- Developing a protocol for quantifying mitochondrial morphology parameters and fluorescence signal intensity.
Main Results:
- The combined TMRM and MG staining allows for simultaneous assessment of mitochondrial morphology and Δψ.
- The protocol is particularly effective in large, flat cells like PHSFs, yielding high mitochondria-specific fluorescence.
- Optimal imaging was achieved at x40 magnification, providing robust data.
Conclusions:
- The described method provides an integrated approach to study mitochondrial morphology and function in living cells.
- This technique is suitable for semiquantitative analysis of mitochondrial membrane potential and morphology in PHSFs.
- The protocol enhances the ability to investigate mitochondrial dynamics and health in cellular research.
