Measuring Mitochondrial Function in Permeabilized Cells Using the Seahorse XF Analyzer or a Clark-Type Oxygen...

Area of Science:

• Cellular metabolism
• Mitochondrial function
• Biochemical assays

Background:

• Mitochondrial respiration is crucial for understanding cellular metabolism and its dysregulation in diseases like cancer, metabolic disorders, immunology, and neurodegeneration.
• Assays with intact cells provide general bioenergetic profiles but lack control over substrate provision, limiting mechanistic understanding.
• Isolation of mitochondria yields poor quality and quantity, often with subselection bias.

Purpose of the Study:

• To present techniques for measuring mitochondrial respiration in permeabilized cells.
• To overcome the limitations of intact cell and isolated mitochondria assays.
• To enable controlled substrate provision for in-depth mechanistic studies of mitochondrial function.

Main Methods:

• Plasma membrane permeabilization of adherent and suspended cells.
• Respiration measurements using Seahorse XF Analyzer for permeabilized adherent cells.
• Respiration measurements using Hansatech Oxygraph for permeabilized suspended cells.

Main Results:

• Permeabilization allows precise control over the experimental medium surrounding mitochondria.
• Enables detailed analysis of mitochondrial bioenergetics with improved substrate control.
• Provides a reliable method for assessing mitochondrial respiration in various cell types.

Conclusions:

• Measuring respiration in permeabilized cells offers a robust solution for studying mitochondrial function.
• This technique enhances mechanistic understanding of metabolism in health and disease.
• It provides a valuable alternative to intact cell and isolated mitochondria assays for bioenergetic profiling.