Preparation of Mitochondria from Mouse Liver

Mitochondria were isolated from female Balb/c mouse liver by a modified version of that described by Brustovetsky, N. & J.M. Dubinsky (2000) J. Neurosci. 20:103.

Materials

  • Phosphate buffered saline (PBS)
  • Buffer A: 225 mM Mannitol, 75 mM Sucrose, 0.1 mM EGTA, 1 mg/mL fatty acid free BSA (Sigma), 10 mM HEPES-KOH (pH 7.4)
  • Buffer B: 225 mM Mannitol, 75 mM Sucrose, 0.1 mM EGTA, 10 mM HEPES-KOH (pH 7.4)
  • Buffer C: 395 mM Sucrose. 0.1 mM EGTA, 10 mM HEPES-KOH (pH 7.4)
  • Buffer D: 1.28 M Sucrose, 0.4 mM EGTA, 40 mM HEPES-KOH (pH 7.4)
  • Tenbroeck ground glass homogenizer, 7 mL (Kontes, VWR Catalog # 85000-07).
  • Dounce homogenizer (VWR Catalog # 885300-007).
  • Sorvall SS34 rotor, Sorvall SA600 rotor or equivalent
  • Centrifuge and rotor capable of 41,000 x g (for enriched protocol)

Methods

Note: Solutions, centrifuge, centrifuge rotor, and centrifuge tubes and homogenizers should be prechilled to 2-8° C. All steps are at 2-8° C unless noted otherwise.

Homogenization of Mouse Liver

  1. Rinse the mouse liver twice with PBS. Homogenize the liver in 5 mL Buffer A/0.5 gram of tissue with ten strokes in a Tenbroeck homogenizer.
  2. Homogenize the resulting slurry with 30 strokes of a tight fitting pestle in a Dounce homogenizer.
  3. Dilute the homogenate with 5mL of Buffer A.

Preparation of Crude Mitochondria

  1. Centrifuge the homogenate for 10 minutes at 600 x g (2200 rpm/Sorvall SS34 rotor; 2100 rpm/Sorvall SA600 rotor). Transfer the supernatant to clean centrifuge tubes and discard the pellet.
  2. Centrifuge the resulting supernatant for 10 minutes at 15,000 x g (11,000 rpm/Sorvall SS34 rotor; 10,100 rpm/Sorvall SA600 rotor). Discard the resulting supernatant.

    A crude mitochondria preparation is typically all that is required for the Cytochrome c Release Assay using recombinant Bcl-2 family members. To use a crude preparation of mitochondria for these assays, proceed to Step 13. To further enrich the mitochondria on a Percoll gradient proceed to step 6.

Preparation of Enriched Mitochondria

Note: Solutions, centrifuge, SW28.1 centrifuge rotor, and centrifuge tubes should be prechilled to 2-8° C.

  1. Resuspend the pellet by repeated pipetting in 10 mL of Buffer B. Centrifuge at 15,000 x g.
  2. Resuspend the pellet by repeated pipetting in 8 mL of Buffer C.
  3. Prepare Percoll-containing solutions by mixing Percoll (100%) with Buffer D according to the following table:
      Percoll Buffer D Deionized Water
    60% Percoll (10 mL) 6 mL 1.5 mL 2.5 mL
    26% Percoll (20 mL) 5.2 mL 3.7 mL 11.1 mL
  4. Add 5 mL of 60% Percoll to the bottom of a centrifuge tube. Carefully overlay with 9 mL of 26% Percoll. Overlay this with 3 mL of the mixture containing resuspended mitochondria in Buffer C (from Step 7 above).
  5. Centrifuge at 41,000 x g (14,600 rpm) for 30 minutes.
  6. After centrifugation, collect mitochondria present at the interface formed between the 26% and 60% Percoll in a volume of 0.5 mL.

    Note: Typically, the protein concentration of the 0.5 mL of mitochondria isolated in a single centrifuge tube is 2.3 mg/mL. The general appearance of the gradient is: a clear top, cloudy material collecting at the uppermost interface, a compact band containing mitochondria at the interface of the 26% and 60% Percoll, and clear 60% Percoll at the bottom.

  7. Use an aliquot of the mitochondria for the Cytochrome c Release Assay.

Preparation of Crude Mitochondria

  1. Resuspend the pellet by repeated pipetting in 1.7 mL of Buffer C. Use this crude preparation for the Cytochrome c Release Assay.

    Note: Mitochondrial preparations should be used within 3 hours.