In vitro cell primary culture and subculture experimental steps (2) precautions

Third, the calculation of experimental results

1. One mouse can obtain (1 to 2.5) x 108 spleen cells.

2. After the cells are inoculated, they can be attached to the wall within a few hours and start to grow. For example, the density of the inoculated cells is appropriate, and a single layer can be formed within 5 days to one week.

3. In general, the cells after passage can be attached to the wall of the culture bottle in about 2 hours. In 2 to 4 days, a single layer can be formed in the bottle, which needs to be subcultured again.

Fourth, matters needing attention

1. The material requirements are fresh, sterile, and when dissecting the mouse, be careful not to damage the spleen and the organs around it, especially the intestines, to prevent contamination of the spleen.

2. When washing the spleen, wash the blood dirt as much as possible, remove the useless tissue, and prevent the tissue from drying out.

3. Rinse the screen with water after grinding to prevent the cells and cells from blocking the mesh.

4. Before counting, pay attention to the cells in the dish and mix thoroughly to disperse the cells into individual cells.

5. The experimental operation should be carried out in the sterile area in the center of the console, and should not be operated in the non-sterile area at the edge.

6. The metal instrument can't be burned in the flame for too long. The burnt metal crucible needs to be cooled before the tissue can be taken to avoid tissue damage.

7. When the rubber plug is over the flame, it can't be long, so as to avoid burning charred gas and harm the cultured cells.

8. After sucking the nutrient solution, the straw can no longer be burned with flame, because the nutrient solution remaining in the pipette head can be charred to form a carbon film, and when used, the harmful substances will be brought into the nutrient solution.

9. Do not touch the mouth of the sterilized vessel, the inside of the stopper, the front part of the straw, etc., which may be in contact with the cells. If it has been touched, use a flame to burn or replace the spare parts.

10. When the cell culture flask is opened or closed, the flame sterilization time is short. Prevent cells from burning out due to excessive temperature.

11. Dump the waste liquid when changing the liquid. The bottle mouth should not touch the waste liquid tank. The speed should not be too fast to prevent the waste liquid from splashing.

12. When blowing the cells, pay attention to whether the cells in the corners are blown down.

13. Hands or relatively dirty items cannot pass through the open bottle opening, ie they cannot be operated above the open container.

14. Only one cell is treated per operation to avoid cross-contamination of cells.

15. Pay attention to your own safety and be especially careful about cell lines from human or viral infections. During the operation, avoid the generation of aerosols, beware of toxic reagents such as DMSO, and avoid sharp objects from injuring people.

16. The cultured cells from the proliferative phase to the formation of dense monolayer cells can be used for cryopreservation, but preferably logarithmic growth phase cells. It is best to change the culture solution one day before cryopreservation.

17. When placing the frozen tube in or out of the liquid nitrogen container, it is necessary to do a protective work to avoid frostbite.

18. It is best to use freshly prepared culture fluid for cryopreservation and resuscitation.

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