Chapter 7: Microsomes

7.1 Lysosome Isolation in Isotonic Sucrose




  1. Decapitate and exsanguinate a rat that has been starved for at least 24 hours prior to the lab. 5

    Fill a syringe with saline and gently perfuse the liver by forcing the saline through the hepatic portal vein, and through the liver.

  2. Remove the liver, place it in a preweighed beaker and weigh the beaker and liver. Calculate the weight of the liver.

  3. Prepare a 10% (w/v) homogenate or brei. For each gram of liver, add 9.0 ml of 0.25 M sucrose in 10 mM Tris-HCL, pH 7.4. to the beaker.

  4. Gently chop the liver in the sucrose and transfer the chopped liver to a teflon homogenizer. 6

    Gently homogenize the liver while keeping it chilled.

  5. Centrifuge the brei at 12,000 xg for 10 minutes at 4 ° C. Decant the supernatant into a chilled beaker and discard the pellet.

  6. Add 0.08 M CaCl_2 to the supernatant to yield a final concentration of 8 mM (use 1 ml of CaCl_2 per 9 ml of supernatant). Stir gently and recentrifuge at 25,000 xg for 15 minutes at 4 ° C

  7. Carefully remove and discard the supernatant. 7

  8. Resuspend the pellet (containing the lysosomes) in 30 ml of 150 mM KCl in 10 mM Tris-HCl Buffer, pH 7.4.

  9. Re-sediment the lysosomes by a final centrifugation at 25,000 xg for 15 minutes at 4 ° C.

  10. Remove a small portion of the pellet for Exercise 7.2. Resuspend the remainder of the pellet in 30 ml of 150 mM KCl/10 mM Tris-HCl Buffer. This suspension is the lysosome fraction for Exercise 7.3.

  11. Prepare a wet mount of the resuspended lysosome pellet and observe with a phase contrast microscope at 100X. Draw any structures observed.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --