Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation

Area of Science:

• Mitochondrial Biology
• Cellular Respiration
• Proteomics

Background:

• Mitochondria are crucial for cellular energy production.
• Efficient isolation of pure, functional mitochondria from skeletal muscle is essential for studying bioenergetics and disease.

Purpose of the Study:

• To establish a Percoll density gradient method for isolating purified mitochondria from mouse skeletal muscle.
• To assess the bioenergetic function and purity of isolated mitochondria.

Main Methods:

• Mitochondria were isolated from mouse quadriceps femoris skeletal muscle using a Percoll density gradient.
• Mitochondrial bioenergetic function was assessed using Seahorse XF Analyses.
• Sample purity was evaluated using mass spectrometry (proteomics).

Main Results:

• The Percoll gradient method yielded minimally contaminated mitochondrial preparations within 3-4 hours.
• Isolated mitochondria demonstrated high functionality, with respiratory control ratios ranging from 3.9 to 7.1.
• Proteomic analysis confirmed significant enrichment of mitochondrial proteins, indicating high sample purity.

Conclusions:

• This study established a reliable methodology for obtaining high-quality mitochondria from mouse skeletal muscle.
• The method ensures sufficient mitochondrial yield for downstream analyses like bioenergetics and proteomics.