The experimental operation should be performed in a sterile area in the center of the operating table, and do not operate in a non-sterile area at the edge. When dissecting mice, be careful not to damage the spleen and its surrounding organs, especially the intestines, to prevent contamination of the spleen. 8. Hands or relatively dirty objects cannot pass through the open mouth, that is, they cannot be operated above the open container. 5. Metal instruments should not be burned in the flame for too long. Prevent burning dead cells due to high temperature. If they are in contact, they should be disinfected with flames or replaced by spare parts. 13. 9. 18. 16. Only one cell line is processed for each operation to avoid cell cross contamination.
One mouse can obtain (1～2. 3. 14.5)×108 spleen cells. Cells can adhere to the wall and start to grow within a few hours after seeding. 4. Do not touch the mouth of the sterilized utensils, the inside of the cork, the front of the straw and all the parts that may be in contact with the cells. 12. When pipetting the cells, pay attention to whether the cells at the corners are pipetted down.. When putting the cryotube into the liquid nitrogen container or taking it out of it, take precautions to avoid frostbite.
Observe the cell morphology under an inverted microscope to determine whether the cells need to be subcultured. 3. 5. (3) Cryopreservation: 1. Remove the mouse and drain the alcohol, put it in a clean bench, and fix it on a foam board. In addition, slow freezing can make the water in the cell seep out of the cell and reduce the formation of ice crystals in the cell.1. Digest at 37°C.25% pancreatin to the culture flask.. Place the sieve in a petri dish, place the spleen on the sieve, use elbow tweezers to hold it, gently grind on the sieve, and constantly drip and rinse with serum-free culture solution. 7.
These two cryo rack can increase the permeability of cell membranes to water.5ml cells). For semi-adherent cultured cells, without pancreatin, pipetting directly, adding fresh medium, and then distributing them into bottles. After the culture medium bottle is opened, pass the flame of the alcohol lamp and place it around the flame of the alcohol lamp.). At present, glycerol or DMSO is often used as a protective agent for cell cryopreservation. The amount should be covered with a thin layer. After adding appropriate culture medium, transfer the cells to a culture flask and incubate at 37°C. (2) Subculture: 1. 4. 4. 2.
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