Introduction
Impaired mitochondrial function has been linked to many diseases, such as stroke, heart disease, cancer, Type II diabetes and Parkinson’s disease. Due to the generation of reactive oxygen and nitrogen species, mitochondria are a major cause of oxidative stress in aging. In order to gain a better understanding of the contributions and interrelations of mitochondrial dysfunction with various diseases, as well as their role in normal development, experiments often need to focus on purified or enriched preparations of mitochondria rather than on intact tissues or whole organisms. Proteomic and metabolomic profiling of mitochondria from various tissues have the potential to provide important insights into mitochondrial function and dysfunction, and may also address fundamental questions of cellular energetics and oxidative stress. As potential drug targets, high quality functional mitochondrial isolates are also important for drug screening studies [1]. Extraction of mitochondria from whole tissues is typically performed by homogenization using labor-intensive manual disruption methods [2] that require extensive operator experience, and often result in damage to fragile organelles and high sample-to-sample variability. Here we describe a semi-automated method that uses a novel gentle mechanical homogenizer (The PCT Shredder, or The Shredder SG3) followed by hydrostatic pressure cycling to release mitochondria from rat tissues with minimal hands-on time (Figure 1). Pressure Cycling Technology (PCT) destabilizes molecular interactions by rapidly and repeatedly raising and lowering pressure in the reaction . At pressures in the 10,000 - 20,000 psi range, PCT can be used to gently lyse cells and release intact organelles, such as mitochondria. This method has been shown to be relatively gentle, and it has already been used to isolate mitochondria from cell culture [3].