Exercise 8.4 - Isolation of Mitochondria

LEVEL I

Materials

• Rat, mouse or suitable source of fresh liver 6
  
• 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5 (Homogenization buffer)
  
• 0.25 M sucrose in 10 mM HEPES, pH 7.5 + 1 mM EDTA (Suspension buffer)
  
• Teflon homogenizer
  
• Refrigerated centrifuge
  
• Janus Green B
  
• Hemacytometer and microscope

Procedure

1. Sacrifice and exsanquinate a rat that has not been fed for at least 24 hours prior to lab.

2. Remove the liver and weigh it.

3. Add the liver to a beaker, and for each gram of liver, add 9.0 ml of 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5. This will produce a 10% brei, a term used to indicate a homogenized suspension.

4. Add the brei to centrifuge tubes and centrifuge at 4,500 xg for 10 minutes at 4° C.

5. Decant the supernatant into clean centrifuge tubes and discard the pellet.

6. Recentrifuge the supernatant at 16,000 xg for 25 minutes at 4° C.

7. Decant and discard the supernatant. Resuspend the pelleted mitochondria in 20 ml of 0.25 M sucrose in 10 mM HEPES. Skip to step 10.

   OR

   Optional: if cleaner mitochondria are desired, resuspend in 20 ml of 0.25 M sucrose in 10 mM HEPES + 1 mM EDTA and perform steps 8 and 9.

8. Recentrifuge the suspended pellet at 16,000 xg for 25 minutes at 4° C.

9. Decant and discard the supernatant. Resuspend the washed pellet in 20 ml of fresh sucrose without EDTA and place the suspension in an ice bath until further use is required. The suspension will remain active for approximately 4-6 hours if kept cold.

10. Mix a few drops of Janus Green B solution with 0.1 ml of mitochondrial suspension. Place one drop of this mixture in a hemocytometer and determine the number of mitochondria per ml. If there are too many mitochondria to count, make serial dilutions of 1/10 to 1/1000 and recount. Diluted mitochondria must be counted rapidly. They are not stable and will decompose if not counted within a few minutes of the dilution.
    Record the number of mitochondria/ml______________