Isolation of Mitochondria from Tissue Culture Cells

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 ∼10 9 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.

MATERIALS

Reagents

Cell pellet derived from 1–5 × 10 9 tissue culture cells

MS 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.

RSB hypo buffer

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

Equipment

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

METHOD

• 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.
  • If a Potter-Elvehjem homogenizer is used in Step 1, then break open the cells with the Teflon pestle rotating at ∼1600 rpm.
  • 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.
  • See Troubleshooting.
  • The top of the pellet will be loose, so be careful not to collect it with the supernatant.
  • This wash is not necessary if a density gradient will be performed (see Protocol: Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation [Clayton and Shadel 2014]).
  • The mitochondria can be stored at −80°C for at least 1 yr for some purposes (e.g., protein isolation).

  TROUBLESHOOTING

  Problem (Step 4): Too many or not enough cells have lysed.

  Solution: Homogenization works best if the cells are resuspended in at least 5–10× the volume of the cell pellet and if the cell suspension occupies at least half the volume of the homogenizer. Homogenization should be performed as quickly as possible because it is performed in a hypotonic buffer. The Dounce homogenizer disrupts swollen tissue culture cells by pressure change. As the pestle is pressed down, pressure around the cell increases. When the cell slips past the end of the pestle, the sudden decrease in pressure causes the cell to rupture. If the pestle is very tight fitting, there may be some mechanical breakage as well. If an excessive number of strokes are needed for good cell breakage, a tighter-fitting homogenizer is needed.

  DISCUSSION

  This basic protocol can be modified to suit special purposes. For example, if the mitochondria are being purified to isolate mitochondrial DNA, contamination with nuclei, not the small organelles, is a problem and the following modifications could be made: Harvest the cells in stationary growth phase when the fewest cells will be actively dividing, substitute CaCl₂ for MgCl₂ in the RSB hypo buffer to stabilize the nuclear membrane, omit washing the mitochondrial pellet, omit any density gradient purification, resuspend and lyse the mitochondrial pellet from Step 10, and purify the mitochondrial DNA from any remaining nuclear DNA (Hudson et al. 1968).

  Footnotes

  • ↵ 4 Correspondence: gerald.shadelyale.edu
  • © 2014 Cold Spring Harbor Laboratory Press