High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models

Area of Science:

• Cell Biology
• Biochemistry
• Proteomics

Background:

• Omics studies reveal links between mitochondrial dysfunction and cellular diseases.
• Validating omics findings requires robust assays for organelle function.
• Mitochondrial membrane potential is a key indicator of mitochondrial activity, but high-throughput measurement methods are lacking.

Purpose of the Study:

• To develop and validate an unbiased, high-throughput imaging-based methodology for quantifying mitochondrial membrane potential.
• To establish a versatile method applicable to diverse in vitro models, from 2D cultures to 3D spheroids.

Main Methods:

• Developed a high-content imaging-based approach for unbiased, high-throughput quantification of mitochondrial membrane potential.
• Applied the method to various models including human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers.
• Integrated automated image analysis and machine learning for cell subpopulation discrimination and analysis in co-cultures.

Main Results:

• Successfully quantified mitochondrial membrane potential across multiple 2D and 3D cell models.
• Demonstrated the method's ability to distinguish between different cell types (melanoma vs. macrophages) in co-culture.
• Validated the method's applicability for large-scale profiling of mitochondrial activity.

Conclusions:

• The developed methodology provides a widely applicable strategy for high-throughput mitochondrial activity profiling.
• This approach facilitates the validation of omics-derived findings related to mitochondrial function.
• Enables advanced analysis of mitochondrial membrane potential in complex cellular models and co-cultures.