Chapter 1 Isolation and subfractionation of mitochondria from animal cells and tissue culture lines

This chapter describes the methods for mitochondrial isolation used in experiments from both human and animal tissues. Further manipulations of isolated mitochondria give the possibility for better investigations of some enzymatic pathways related to the mitochondrial function. The initial destruction of intercellular connections, cell walls, and plasma membranes is necessary for mitochondrial isolation. The choice of a destruction technique should be dictated by the type of cells and intercellular connections, and is either mechanical or enzymatic. The ratio of tissue to isolation medium is usually 1:5 to 1:10 (w/v); the use of proteases softens the tissue, especially muscle, prior to homogenization. The homogenization step is always carried out on ice in order to inactivate cellular enzymes that could damage mitochondria. The nature of the contaminants that may be present in a mitochondrial preparation depends on the biological material from which they are isolated. Except for erythrocytes, few cells are small enough to escape sedimentation during the low-speed centrifugation. However, it is possible to find pinched-off cell fragments; these can be eliminated by gradient centrifugation.