Mitochondrial extraction and observation

mitochondrials are important cytocytes in cells and are present in most living cells, and their primary function is to provide the energy needed to metabolize various substances in the cells. Because of this, the study of the structure, function and physicochemical properties of components such as mitochondrial membranes, respiratory chain enzymes and mitochondrial
DNA
has become an important topic in the study of
cellular biology
In cells of organs such as kidneys and
tissues
, a large number of mitochondrials are extracted from these organ tissues, and when there are fewer samples used (e.g. observations of electronscopes and photonscopes) can be extracted from tissue
cultured
cells, this experiment is to introduce two materials prepared for photoscopic observation of mitochondrials.
, purpose and requirements areTo understand the basic principles and processes of extracting mitochondrials, through the observation of optical
scope
to understand the general form of mitochondrial separation outside the body
2, basic principles
mitochondrials have a complete structure, a certain size and mass, at low temperatures in iso-permeable cells, differential centrifugation, to obtain mitochondrials. After
the active dye
Janus green B staining, mitochondrials are light blue.
third, the experimental content1. Mitochondrial separation extract 2. The homogeneity preparation of rat liver 3. The living chromosomes of mitochondrials
4, experimental steps(i) the separation of mitochondrials in animal tissue, extraction and observation
1. Microscope examination: 1% Janus green B solution is added to mitochondrial suspension at a 1:1 ratio, dyed at room temperature or in a water bath for 15 to 20 minutes, with
straw
to absorb a drop of mitochondrial suspension, drop on the slide, cover the slide, under the microscope to observe, mitochondrial blue-green round particles.
2. The extraction and observation of mitochondrials of tissue cultured cells
(iii) should be paid attention to in
1. In order to ensure the integrity of mitochondrials, the operating environment should be kept constant as much as possible, such as temperature (0-4 degrees C), pH (around 7.0), while keeping the operating time as short as possible.
2. The tissue cells should be particularly careful when digesting to prevent loss or repetition. (Loss refers to the loss of cells into the digestive fluid).
3. When homogenized, the medium used must be an iso-permeable buffer, commonly used in 0.25 mol/L sucrose solution or physiological saline instead of Hank's liquid
4. The number of homogenizers depends on the tightness of the homogenizer, the number of times is too small, cell damage is incomplete, will affect mitochondrial yield.
5. So take 2/3 of the night used to prepare mitochondrials is to prevent excessive cell fragments from affecting observation.
6. The whole separation process, generally best in 30-60 minutes to complete, should not be too long.
, exercises,1. Describes the extraction process of mitochondrials in animal cells and their considerations.
2. How to identify the final product extracted by the above method is mitochondrials?
。