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. Analyt Biochem 418:213-23. |
Gross VS, Greenberg HK, Baranov SV, Carlson GM, Stavrovskaya IG, Lazarev AV, Kristal BS (2011) Analyt Biochem
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
• O2k-Network Lab: US_MA Boston_Kristal BS, US_PA Pittsburgh_Baranov SV
Labels:
Organism: Rat
Tissue;cell: Skeletal muscle, Kidney
Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property.
Coupling state: OXPHOS
HRR: Oxygraph-2k
PBI-Shredder
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: http://www.oroboros.at/index.php?shredder
