What Is the Kinetochore?

Mitochondria (mitochondrion) ^[1] is an organelle that is covered by two layers of membranes in most cells. It is a structure that produces energy in cells. It is the main place for cells to perform aerobic respiration. It is called "power house" . Its diameter is around 0.5 to 1.0 microns.

There are two hypotheses for the origin of mitochondria, namely

Mitochondria are one of the most sensitive organelles to various injuries. The most common pathological changes during cell injury can be summarized as changes in the number, size, and structure of mitochondria:

Mitochondria are the parts that use oxygen to make energy directly. More than 90% of the oxygen inhaled into the body is consumed by mitochondria. However, oxygen is a "double-edged sword".

Problems with human mitochondria can cause mitochondrial disease, which is a large category

Mitochondrial staining

Mitochondria-Teach: Tablet # 3

Puppy pancreas, Regaud's solution fixed, paraffin section, iron hematoxylin staining.

The mitochondria are black stained with iron hematoxylin and are distributed in the cytoplasm around the nucleus. The mitochondria are granular, linear or short rod-shaped, or straight or curved under a high-power microscope, with a clear outline.

The secretory cells of the pancreas are conical, with large and round nuclei, located in the center of the cell, and many large, round black particles are gathered at the free end of the cell as secretory particles.

Observation of mitochondria extraction

Mitochondria are important organelles in cells and exist in most living cells. Its main function is to provide the energy required for the metabolism of various substances in the cell. Because of this, the study of the structure, function, and physicochemical properties of mitochondrial membranes, respiratory chain enzymes, and mitochondrial DNA has become an important subject in cell biology research. Therefore, the technology of extracting mitochondria has become essential in mitochondrial research. A large number of mitochondria are present in cells with strong metabolism, such as the cells of animals' heart muscle, liver, kidney and other organs and tissues. A large number of mitochondria are extracted from these organs and tissues. Microscopic observation) can be extracted from tissue culture cells. This experiment is to introduce two materials to prepare mitochondria for light microscope observation.

I. Purpose and requirements

Understand the basic principle and process of mitochondrial extraction, and understand the general morphology of mitochondria isolated in vitro through observation with optical microscope.

Second, the basic principle

Mitochondria have a complete structure, a certain size and quality. Cells are broken in isotonic fluid under low temperature conditions, and mitochondria are obtained after differential centrifugation. The mitochondria were light blue when stained with Janus green B, a reactive dye.

Experimental content

1. Isolation and extraction of mitochondria 2. Preparation of homogenate of rat liver 3. Live staining of mitochondria

Fourth, the experimental steps

(I) Isolation, extraction and observation of mitochondria in animal tissues

Microscopy: Add 1% Janus green B solution to the mitochondrial suspension in a 1: 1 ratio, and dye for 15-20 minutes at room temperature or in a water bath. Pipette a drop of mitochondrial suspension with a pipette, drop it on a glass slide, and cover the slide Then, it was observed under a microscope, and the mitochondria were blue-green circular particles.

2. Extraction and Observation of Mitochondria in Tissue Culture Cells

Mitochondrial attention issues

1. In order to ensure the integrity of the mitochondria during the entire operation, the environment (such as temperature (0-4 ° C)) and pH (about 7.0) should be kept constant during the operation, and the operation time should be as short as possible.

2. Care should be taken in the digestion of tissue culture cells to prevent loss or repetition. (Loss refers to the shedding of cells into the digestive juices).

3 When homogenizing, the medium used must be an isotonic buffer solution. A 0.25 mol / L sucrose solution or physiological saline is often used instead of Hank's solution.

4 The number of homogenizations is determined by the tightness of the homogenizer. If the number of homogenizations is too small, the cell damage is incomplete, which will affect the mitochondrial production.

5. Therefore, 2/3 of the supernatant was used to prepare mitochondria to prevent excessive cell debris from affecting the observation.

6. The entire separation process is generally best completed within 30-60 minutes, and should not be too long.