Base Reference: Kristian et al, J. Neurosc. Methods 152:136‐143 (2006)
- Homogenization buffer (HB): 210mM mannitol, 70mM sucrose, 5mM HEPES pH 7.12 at 25oC. Works fine using HEPES diluted from 1M stock (Gibco cat # 15630)
- Homogenization buffer supplemented (HBS) with EGTA at a final concentration of 1mM and Roche’s Complete Mini EDTA‐free protease inhibitor cocktail (cat # 11 836 170 001) at a final concentration of 1X (1 tablet dissolved in 10ml of buffer)
- 50% Percoll: Percoll *(Sigma cat # P1644) diluted 1 to 1 with 2X HB.
- 22% and 15% Percoll solutions: The 50% Percoll stock is used to prepare all other Percoll solutions; to that effect dilutions of the 50% Percoll solution are done in HBS.
- EGTA 1M pH 7.5 stock
*Sigma Percoll solution is considered to be a 100% solution
- Grow cells to approx 85% confluence in media of choice. For N2a cells the procedures are described for cells grown in 4 tissue culture plates with a diameter of 15cm2.
- Transfer plates to a bed of ice
- Wash each plate twice with 8 ml of ice cold HB.
- Collect the cells by scraping with a cell culture policemen using 1ml of HBS per plate. Repeat the collection once more (some times it is necessary to conduct a third scraping, depending of how well the cells adhere to their substrate. A simple visual inspection indicates if a third collection is necessary). Combine all collected cells. It is best to collect directly in the Potter homogenizer.
- Homogenizer using a Potter Homogenizer whose pestle is driven at an estimated 2400 rpm by a GT motor control (Glass‐Col, Terre Haute, IN) at 40% capacity and a Glass‐Col Continue Duty DC motor (cat # 099C_K44). Use 20 strokes of the pestle in a period of 3 min, maintaining the homogenizer tube in ice. Repeat once.
- Spin the homogenate at 1,500g for 3 min
- Collect the supernatant and spin it at 13,000g for 17 min.
- Resuspend the pellet in 1.4 ml of HBS
- Prepare two 5ml centrifuge tubes with a discontinued Percoll gradient as follows: 1ml of 50% Percoll solution as cushion followed by layering 3ml of a 22% Percoll solution.
- To the resuspended pellet add 0.3 ml of 50% Percoll (final sample should have a concentration of Percoll of 15%.) and layer 1ml of the 15% Percoll sample on top of the described 50%‐22% gradient. Use 15% Percoll to help balance the centrifuge tubes before centrifugation.
- Spin at 30,700g for 6min.
- Recover the mitochondria from the 50% ‐ 22% Percoll interface
- Wash the mitochondria by diluting 1 to 10 with HBS at 15,600g for 30 min. Repeat the wash step to decrease the amount of percoll, protease inhibitors and EGTA to 0.25X, or use the buffer of choice depending on the needs of the experiment at hand.
Address question and/or comments to
Mirta Mihovilovic, Ph.D.
Duke University Medical Center