Isolation of Mitochondria from Tissue Culture Cells

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.



Cell pellet derived from 1–5 × 109 tissue culture cells

cautioncautionMS homogenization buffer (1× and 2.5×)

MS homogenization buffer is an iso-osmotic buffer used to maintain the tonicity of the organelles and prevent agglutination.

cautionRSB hypo buffer

RSB is a hypotonic buffer used for swelling tissue culture cells.


Centrifuge tubes

Dounce homogenizer (15 mL) with a tight-fitting B pestle or Potter-Elvehjem homogenizer (5 mL) with a Teflon pestle (see Steps 1 and 3)

Phase contrast microscope


The solutions, tubes, and homogenizer should be prechilled on ice. All centrifugation steps are at 4°C.

  1. Resuspend the cell pellet in 11 mL of ice-cold RSB hypo buffer and transfer the cells to a 15-mL Dounce homogenizer.

Alternatively, as described by Frezza et al. (2007), resuspend the cell pellet in 9 mL of ice-cold RSB hypo buffer and transfer 3 mL of the cells at a time to a 5-mL Potter-Elvehjem homogenizer with a Teflon pestle.

  1. Allow the cells to swell for 5–10 min. Check the progress of the swelling using a phase-contrast microscope.
  2. Break open the swollen cells with several strokes of the B pestle. For each stroke, press the pestle straight down the tube, maintaining a firm, steady pressure.

If a Potter-Elvehjem homogenizer is used in Step 1, then break open the cells with the Teflon pestle rotating at ∼1600 rpm.

  1. Check the degree of homogenization with a phase-contrast microscope.

Naked nuclei (smooth spheres with obvious nucleoli inside), smaller organelles (dark, granular objects), and a small number of unbroken cells (large spheres with a granular appearance) should be present if cell lysis was successful. Eight to nine naked nuclei for every whole cell is a very good result. Trying for anything better usually results in increasing the number of damaged nuclei, which increases the number of mitochondria trapped in the nuclear pellet during the first centrifugation.


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