Gross 2011 Anal Biochem
|Gross VS, Greenberg HK, Baranov SV, Carlson GM, Stavrovskaya IG, Lazarev AV, Kristal BS (2011) Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization. Anal Biochem 418:213-23.|
Abstract: Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes and cancer. There is growing recognition of the importance of mitochondria as both targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible, high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semi-automated methods to disrupt tissue, using kidney and muscle mitochondria preparations as exemplars. These methods utilize either a Barocycler, or The PCT Shredder, or both. The PCT-Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of over-homogenization. Mitochondria isolated using The PCT-Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of The PCT-Shredder and Barocycler to be used to purify mitochondrial preparations. • Keywords: Mitochondria, Barocycler, Hydrostatic Pressure, Kidney, Muscle, Drug discovery, Toxicology, PCT-Shredder, Oxygraph-2k
Organism: Rat Tissue;cell: Skeletal muscle, Kidney Preparation: Isolated mitochondria
Coupling state: OXPHOS
Conclusion: "The new methods require little hands-on tissue homogenization, can be easily learned by a novice, and are expected to lead to results that are more consistent and reproducible from lab to lab."
- More information: PBI-Shredder O2k-Set