Isolation of mitochondria

Due to the diversity of the tissues involved and too many variations, it is difficult to provide specific speeds, times or procedures. Here are just a few suggested procedures.

• Two-step homogenization using first the KINEMATICA POLYTRON^® homogenizer followed by a precooled Teflon^® mortar and pestle technique (Potter Method).
• Homogenize with several short pulses of a POLYTRON tissue homogenizer (for example, three 15-s periods) using approximately 2 ml buffer per gram of tissue.
• Homogenize suspension with a loose-fitting Teflon pestle and then with a tight-fitting Teflon pestle (four strokes each).
• Thoroughly homogenize with 20 strokes at 800 rpm on ice by using a motor-driven Potter Dounce homogenizer and then centrifuge at 5,600 g for 3 min at 4°C. Keep the supernatant and dissolve the pellet in a mitochondrial medium buffer and homogenize as described above.
• Mince the liver, rinse to remove the blood, and transfer it to 20 ml of HIM buffer + BSA + protease inhibitors in a 50 ml tube. Conduct all subsequent steps at 4°C. Homogenize the liver with a POLYTRON homogenizer for four 1-s periods at a setting of 6.5. Pellet nuclei and unbroken cells at 3,000 rpm for 10 min. Collect the supernatant and keep on ice. Resuspend the pellet in another volume of HIM buffer + BSA and again subject it to a second round of homogenization. Combine and centrifuge both supernatants.
• After being minced finely with scissors, homogenize diaphragm pieces for two 10-s periods by using a POLYTRON homogenizer set at one-half speed.

For more information, do a Web search for “isolation of mitochondria using a POLYTRON.”

Teflon^® is a registered trademark of E.I. Du Pont De Nemours and Company Corp.