Isolation of mitochondria from cells and tissues

. Author manuscript; available in PMC: 2021 Oct 21.

Published in final edited form as: Methods Cell Biol. 2019 Dec 10;155:3–31. doi: 10.1016/bs.mcb.2019.10.002

Problem	Solution
Low yield	Homogenization has a large impact on yield of the mitochondrial preparation. We recommend use of a teflon-glass homogenizer. Homogenization using glass-glass homogenizer can lead to increased yield but can also damage mitochondria. Visualization of cell disruption using a microscope can be used to optimize homogenization conditions for different cell lines. The homogenization step should lyse about 80% of the cells. Fibroblast cell membranes are difficult to break open. To promote homogenization, freeze-thaw cells once harvested.
Low quality	Homogenization and all the subsequent steps of the protocol must be performed at 4°C to minimize the activation of phospholipases and proteases. Excessive homogenization can cause damage to the mitochondrial membrane and trigger release of mitochondrial components. Avoid diluting the mitochondria with buffer. Mitochondria retain their function for a longer period, probably as a result of less exposure to oxygen, when stored in a concentrated form (30–50 mg/mL).

Problem

Solution

Low yield

Homogenization has a large impact on yield of the mitochondrial preparation.
We recommend use of a teflon-glass homogenizer. Homogenization using glass-glass homogenizer can lead to increased yield but can also damage mitochondria.
Visualization of cell disruption using a microscope can be used to optimize homogenization conditions for different cell lines. The homogenization step should lyse about 80% of the cells.
Fibroblast cell membranes are difficult to break open. To promote homogenization, freeze-thaw cells once harvested.

Low quality

Homogenization and all the subsequent steps of the protocol must be performed at 4°C to minimize the activation of phospholipases and proteases.
Excessive homogenization can cause damage to the mitochondrial membrane and trigger release of mitochondrial components.
Avoid diluting the mitochondria with buffer. Mitochondria retain their function for a longer period, probably as a result of less exposure to oxygen, when stored in a concentrated form (30–50 mg/mL).