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Updated: Apr 23, 2026
Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Isolation of mitochondria from tissue culture cells
David A Clayton1, Gerald S Shadel2
1Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147-2408;
Abstract:
The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼10⁹ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.
Insights
This protocol details isolating mitochondria from cultured cells. It involves cell swelling, homogenization, and differential centrifugation to obtain pure mitochondria for research.
Area of Science:
- Cell Biology
- Mitochondrial Research
Background:
- Mitochondrial quantity varies significantly between cell types.
- Human HeLa cells possess more mitochondria than mouse L cells.
Purpose of the Study:
- To outline a protocol for isolating mitochondria from cell cultures.
- To provide a reproducible method for obtaining pure mitochondria.
Main Methods:
- Starting with a cell pellet (∼10⁹ cells).
- Swelling cells in a hypotonic buffer.
- Rupturing cells using homogenization (Dounce or Potter-Elvehjem).
- Isolating mitochondria via differential centrifugation.
Main Results:
- Successful isolation of mitochondria from cell cultures.
- The protocol yields a pure mitochondrial fraction.
Conclusions:
- This method provides a reliable way to isolate mitochondria.
- The isolated mitochondria can be used for further biochemical and cellular studies.
