& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Liver is a convenient source for functional intact mitochondria for a number of reasons. Animal tissue is more readily homogenized than plant tissue because there are no cell walls, and liver in particular is a soft and fairly homogeneous tissue. The metabolism of endotherms requires that some tissues maintain a high density of mitochondria, so the potential yield is high. Isolating mitochondria from highly structured animal tissues such as muscle can be technically difficult since a high proportion of the organelles remain trapped in cell and tissue fragments (although muscle can be a good source). One can quickly and easily obtain a substantial quantity of liver mitochondria within less than an hour's preparation time.
We use Sprague-Dawley albino male rats (Charles Rivers Laboratories, Wilmington, MA) for our studies. Female rats can be used, however for serious studies we usually stick to males to avoid complications due to the estrous cycle. A good weight range for the animal is 200 - 250 gms, perhaps a bit larger. A liver from this size animal yields up to two ml or so of concentrated mitochondria, enough for dozens of oxygraph experiments. In the teaching lab, two sets of lab partners share one animal, dividing the liver tissue into equal starting amounts by weight. Since the liver is responsible for detoxification processes including metabolism of anesthetic agents, and because both an anesthetic and its metabolites can affect liver function, it is preferable from an experimental viewpoint to avoid using anesthetic or tranquilizing drugs. We sedate the animals using isoflurane then decapitate them using a rat guillotine. Isoflurane is short acting and has not appeared to compromise mitochondria function.
The adjective "medial" is an anatomical term meaning vertical, up the middle. We open up the animal with a medial incision (not a "medical" incision) from groin to sternum. We first separate the skin then the underlying muscle and peritoneum, revealing the liver. The liver is brown, large, and almost unmistakable. If the student gets into the rat quickly (opened within 2-3 min of decapitation) and cuts through the sternum, opening the chest, the heart can be seen still beating. The liver should be chilled immediately by pouring a generous amount (100 ml) of ice-cold 0.85% NaCl into the peritoneal cavity. It can be removed in pieces or removed intact by cuting it off at the base, and then it should be dropped into a second beaker of ice-cold saline solution to continue to reduce the temperature. Further steps in the isolation should all be done at ice-bucket temperature.
We find it convenient to divide the tissue from one 200-225 gm rat into two equal portions, each weighing 3 to 5 grams. The ratio of homogenizing medium to tissue is important, as is the depth of liquid in the container when using a shearing type homogenizer. The specific instrument we use is a "Tissuemizer" with T25 stainless steel shaft (Tekmar, Inc., Cincinnatti, OH). A teflon-on-glass (Potter-Elvehjem) homogenizer can also be used, but it takes longer. A shearing type homogenizer is fast but prolonged or overly vigorous homogenization can be damaging to mitochondria. Both methods yield good preparations from rat liver.We have had great success by draining the tissue then mincing it in a 50 ml plastic disposable beaker, followed by addition of 20 ml homogenizing medium (0.25M sucrose, 5 mM HEPES buffer, and 1 mM EDTA, pH 7.2). After mixing to suspend the mince we homogenize at a setting of '40' for 10 sec.
To remove large cell and tissue fragments and cell nuclei (the 'nuclear pellet'), we centrifuge the homogenate at 500 x g for 10 minutes. Before centrifugation, we top off the homogenate with medium to fill each tube. To bring down the mitochondrial pellet we pour the supernatant into a clean centrifuge tube, and without topping off we centrifuge at 9400 x g for 10 min.
When the supernatant is poured off, the loose upper part of the mitochondrial pellet may come off as well. Intact mitochondria tend to sediment more quickly than damaged mitochondria. The loose part of the pellet most likely contains a high proportion of damaged (uncoupled) mitochondria, and can be lost. The white foamy material near the top of the tube consists of lipids, which must be kept from contact with the mitochondria. They can be removed by wiping the inside of the tube with a lab wiper. Mixing of lipids with the mitochondria suspension will cause some degree of uncoupling (loss of ability to maintain respiratory control). After using a pasteur pipet to remove the last bit of liquid, a glass rod should be used to stir the remaining pellet into a smooth paste. We don't add buffer at all. The more liquid that remains with the pellet, the more difficult it is to homogenize all of the particles.We keep the centrifuge tube on ice while stirring, and try not to introduce air into the suspension.
Mitochondria keep best when concentrated, to minimize exposure to oxygen. They remain dormant until diluted into an oxygen-rich respiration medium. The paste should be transferred to an eppendorf tube, and air bubbles avoided by careful pipetting with a micropipettor set to, say, 100 µl or so. The suspensions are very viscous. Care must be taken to allow pressure to equalize after drawing up suspension, otherwise it may shoot up into the pipettor. Use of a pasteur pipet at this point results in loss of much of the pellet, since the material readily sticks to glass. Once transferred to an eppendorf tube the preparation (which must be stored on ice) is ready for use.