Related Experiment Video
Updated: Jun 10, 2026
Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI
Kristian H R Jensen1, Jens C Rekling
1Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
Abstract:
Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine, which is a suspected mitochondrial toxicant. CCCP and DNP have short-term depolarizing effects, and thioridazine has long-term hyperpolarizing effects on the mitochondrial membrane potential of Chinese hamster ovary (CHO) cells. The assay also detected changes of the mitochondrial membrane potential in CHO cells exposed to cobalt (mimicking hypoxia) and in PC12 cells exposed to amyloid β, demonstrating that the assay can be used in cellular models of hypoxia and Alzheimer's disease. The assay needs no washing steps, has a Z' value >0.5, can be used on standard fluorometers, has good post liquid-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction.
Insights
Researchers developed a simple, no-wash assay to measure mitochondrial membrane potential using DASPEI. This tool aids in toxicological testing and discovering drugs for diseases linked to mitochondrial dysfunction.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Mitochondrial dysfunction is implicated in numerous diseases and drug toxicities.
- Mitochondrial membrane potential serves as a key indicator of mitochondrial health.
- Existing assays can be cumbersome and time-consuming.
Purpose of the Study:
- To develop and validate a novel, no-wash assay for measuring mitochondrial membrane potential.
- To assess the utility of the assay in various cellular models and drug screening.
- To establish a rapid and reliable method for evaluating mitochondrial function.
Main Methods:
- Utilized 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI) as a potentiometric probe.
- Developed a 96-well plate assay compatible with standard fluorescent plate readers.
- Validated the assay using protonophores (CCCP, DNP), thioridazine, cobalt (hypoxia model), and amyloid-β (Alzheimer's model).
Main Results:
- The DASPEI assay successfully detected both short-term depolarization and long-term hyperpolarization of mitochondrial membrane potential.
- The assay demonstrated sensitivity in cellular models of hypoxia and Alzheimer's disease.
- Achieved a Z' value >0.5, indicating suitability for high-throughput screening.
Conclusions:
- The no-wash DASPEI assay is a simple, rapid, and robust method for assessing mitochondrial membrane potential.
- The assay is suitable for large-scale screening, toxicological testing, and drug discovery.
- This assay can be valuable for studying diseases associated with mitochondrial dysfunction.
