The purpose of this experiment is to understand the general morphology and growth state of animal cells in culture, and at the same time, you can master the basic method of cell counting. Content source: Laboratory instruction manual of China Medical University.
Operation method
indicator method
Principle
There are two main states of cells cultured in vitro. One is the cells that can be adhered to the culture support, such as HeLa cells, NIH3T3, etc., called wall-adherent cells, and most of the cells cultured in vitro belong to this kind of cells. Another type of cell does not attach to the wall of the container, but is suspended in the culture medium, such as HL60 cells, called suspension type cells, these cells are mainly hematogenous or oncogenic cells. In cell biology experiments, often have to carry out the identification of living cells and cell counting, adjusting the density of cells, is to carry out experiments indispensable to a basic skill.
Materials and Instruments
HeLa Cells NIH3T3 HL60 Cells Move I. Observation of the morphology of cultured cells For more product details, please visit Aladdin Scientific website.
Trypsin EDTA Mixed Digestive Solution
Inverted Microscope Cell Counting Plate General Optical Microscope Nipple Pipette Cover Sheet
1. Take out the cell culture bottle from 37 ℃ carbon dioxide incubator (or warm box), the bottle mouth is slightly upward. Tilt the bottle so that the liquid in the bottle does not touch the stopper or flow out of the bottle, and observe the color and clarity of the cell culture liquid. Observe the color and clarity of the cell culture solution. Then, place the cell culture flask smoothly on the inverted microscope carrier stage. 2.
2. Turn on the light source of the inverted microscope, observe with the 10X objective lens, adjust the field of view through the binocular eyepieces to the appropriate brightness, and adjust the angle of the binocular eyepieces. Adjust the distance between the pupils of the binoculars so that the left and right visual field images are combined into one. 3. Adjust the height of the stage to make a smooth and steady movement of the bottle on the inverted microscope stage.
3. Adjust the height of the stage for focusing, after seeing the cell layer, use the fine adjuster to make the object image clear. After seeing the cell layer, use the fine adjuster to make the object image clear, and pay attention to the outline, shape and internal structure of the cell. The ten times objective lens is often used for this purpose. The objective lens is often used for observation.
4. Results
Adherent cells are generally of two types, epithelial and fibroblastic. Epithelial epithelial cells are flat and irregularly polygonal, with a round nucleus in the center, and are often closely connected to each other as a monolayer, as in the case of Hevea. The epithelial cells are flat and irregularly polygonal, with a round nucleus in the center, and are often tightly connected to each other when growing, such as HeLa cells. Fibroblast-type cells are similar in morphology to fibroblasts in vivo. The morphology of fibroblast-type cells is similar to that of fibroblasts in vivo, with a pike-shaped or irregularly triangular cytosol, an oval nucleus in the center, and two or three cytoplasmic The cytoplasm extends outward with two to three protrusions of different lengths, and the cell groups are often connected into a network by protoplasmic protrusions, and the cells tend to radiate or swirl as they grow. The cell groups are often connected by protoplasmic protrusions to form a network, and the cells tend to be radial or swirling when growing, such as NIH3T3. When adherent cells grow well, they are homogeneous and transparent, with few intracellular particles and no vacuoles. vacuoles, cell edges are clear, suspended cells and debris cannot be seen in the medium, and the culture medium is clear and transparent. Transparent, but when there are more intracellular particles, poor transparency, more vacuoles, and a greater contrast in cell outline enhancement, it indicates that the cell is in poor functional status, and the growth is poor. When there are more particles in the cells, poor transparency, more vacuoles and greater contrast in the enhancement of the cell outline, it indicates that the cells are in poor functional status and poor growth. When the medium in the culture bottle is turbid, we should think of the possibility of bacterial and fungal contamination. When the medium in the culture flask is turbid, the possibility of bacterial and fungal contamination should be thought of. When the edge of suspended cells is transparent and bright, the growth is good; on the contrary, it is poor or dead. The opposite is true, the growth is poor or dead. Because of the presence of PH indicator in the medium, its color can often indirectly indicate the growth status of the cells. indirectly indicate the growth status of the cells. When the color is orange, the cell growth is generally good; when the color is light yellow, the cell growth may be due to the incubation time. yellow color, the culture time is too long, insufficient nutrients, too many dead cells; such as purple-red, the If the color is purplish red, the cells may be in bad growth state or dead. In fact, the morphology of a cell in culture In fact, the morphology of a cell in culture is not constant, it changes with the nutrition, PH, growth cycle, but under relatively stable conditions, the morphology is basically the same. It changes with nutrition, PH, and growth cycle, but under stable conditions, the morphology is basically the same. In the culture of adherent cells, the cells with high refractive index under the microscope, round and bright are generally regarded as dividing cells. In the culture of adherent cells, the cells with high refractive index, round and bright under the microscope are generally regarded as dividing cells. Tumor cells are characterized by overlapping growth.
Counting of cultured cells and identification of living cells.
1. Prepare the counting plate
Clean the counting plate with 95% alcohol and put the cover sheet on top of the counting plate. 2.
2. Preparation of cell suspension
Pour the culture solution from the culture flask into a clean test tube and add 0.25% trypsin-0.02% EDTA to the flask. Add 0.25% trypsin-0.02% EDTA mixed digest 1 ml into the culture flask, let it stand for 3~5 minutes, and wait until the cells become round and not connected with each other. The cells should be left for 3~5 minutes until they become rounded and not connected to each other. Pour the culture solution from the test tube back into the culture flask and blow gently to make cell suspension. Make cell suspension. 3. Staining
Take 0.5 ml of cell suspension, add 0.5 ml of 0.3% Tepan Blue Staining Solution, mix and stain for 3~5 minutes. Mix and stain for 3~5 minutes. 4. 4. Dropwise addition of suspension
After shaking the suspension well, slowly add a small amount of stained cell suspension along the edge of the cover slip onto the slanted surface of the cell counting plate. The liquid should naturally fill the whole counting chamber. If there are air bubbles in the chamber or too much liquid causes the cover slip to drift, redrop the liquid. If there are air bubbles in the chamber or too much liquid to make the cover slip drift, add the liquid again. 5. Counting
Count the number of cells in the four compartments under the 10X objective of an ordinary light microscope. Count the number of cells in the four compartments under the 10X objective of a normal light microscope. 6. Results
Calculate the cell concentration according to the following formula:
Total number of cells in the cell compartments × 104 ① × dilution = number of cells/ml suspension Total number of cells in the cell compartment - number of stained cells × 104 ② dilution factor = number of live cells/ml suspension Note ① Since the volume of each large cell in the four compartments is 0.1 mm3, 1 ml = 10,000 large cells, therefore, 1 cell number×10 4 = number of cells/ml. Note ② Stained specimens should be counted within 15 minutes, because the Taipan-orchid staining solution rapidly stains dead cells and delays the counting time. Delaying the counting time will cause the live cells to be stained as well. Cell counting should be done accurately, therefore, in scientific research, both sides of the counting plate are often added to the cell suspension, and the droplets are counted at the same time. Therefore, in scientific research, it is often added to both sides of the counting plate, and at the same time drop several counting plates (or repeatedly drop a plate several times), and finally take the average of the count. The average of the counts is taken.