Aims
As the major energy source for mammalian cells, mitochondria have been the subject of numerous studies. However, the isolation and purification of healthy mitochondria, especially from fresh tissue, remains challenging. The most popular
Background and aims
As the major energy source for mammalian cells, mitochondria have been the subject of numerous studies. However, the isolation and purification of healthy mitochondria, especially from fresh tissue, remains challenging. The most popular
Conclusions
A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified, uniform and functional mitochondria.
Methods
Fresh mouse liver was homogenized, and cells lysed. Particle size analysis, quantitation of mitochondrial DNA, mitochondrial oxygen consumption, and purity of mitochondria (by electron microscopy) were measured in samples after various purification steps and significant differences determined.
Results
A two-step procedure consisting of centrifugation followed by filtration through 1.2μ and 0.8μ filters resulted in uniform mitochondrial preparations with diameters between 520-540 nm, and approximately 5-times more pure samples. The mitochondria thus obtained had oxygen consumption and sensitivities to mitochondrial inhibitors that were indistinguishable from those purified by centrifugation alone. Electron microscopy confirmed the presence of more uniform and 4-5 times greater concentrations of mitochondria compared to centrifugation alone. Conclusions: A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified, uniform and functional mitochondria.