Inoculation of subcutaneous tumor transplants

　　1. Cell seeding:

　　1.1 Take out the container containing the cell suspension from the ice box, and slowly shake it back and forth to mix the cell suspension thoroughly;

　　1.2 Use a disposable 1ml sterile syringe to absorb an appropriate amount of cell suspension (if each animal is injected with 0.1ml, the amount for 5 animals at a time is 0.5ml), cover the container containing the cell suspension and continue to place it in the Wait in the ice box for the next vaccination;

　　1.3 Grab the mouse with your left hand, expose the skin on the right side of the animal, and disinfect it with a 75% alcohol cotton ball (disinfection must take the needle entry point as the origin and expand to the surroundings). Prick the syringe needle upward from the lower costal margin of the right back of the mouse. When inserting, the needle moves forward close to the lower layer of the skin. During the movement, ensure that the needle is in the subcutaneous area. When the needle tip reaches the back of the right shoulder, the cell suspension is injected subcutaneously into the animal. When withdrawing the syringe, rotate the needle 360 degrees so that the bevel of the needle tip is close to the skin;

　　1.4 After inoculation, when placing the mice back in the cage, observe whether there are any abnormalities in the mice. If there are no abnormalities, continue to inoculate;

　　1.5 Add an appropriate amount of 84 disinfectant solution to the remaining cell suspension after inoculation (the volume of 84 disinfectant solution is not less than 1/10 of the volume of the cell suspension) and discard it together with the container in a medical waste bucket;

　　1.6 Discard used syringes in the sharps container.

　　2. Tumor mass inoculation

　　2.1 Preparation of experimental animals: Take out the experimental animals to be inoculated with tumor cells, and remove the coat in the inoculated area as required;

　　2.2 Preparation of donor animals: Select tumor-bearing mice with good growth status, no tumor ulceration, and tumor volume of 500-800mm3 as donor animals, and euthanize the tumor-bearing mice with carbon dioxide/cervical dislocation;

　　2.3 Preparation of tumor mass: Take the euthanized tumor-bearing mice, disinfect the skin at the tumor site with 75% alcohol cotton balls, and remove the tumor tissue;

　　2.4 Remove the capsule, blood vessels and necrotic tissue surrounding the tumor tissue, cut the tumor, and select the fish-like tissue. Cut the tumor into moderately sized pieces (approximately 3mm × 3mm × 3mm) and place them in a culture dish containing serum-free culture medium. Ice cubes should be placed under the culture dish to ensure the vitality of tumor cells;

　　2.5 Inoculation of tumor mass: Place the tumor tissue in a sterile trocar. The operator grasps the animal with his left hand and exposes the right half of the skin of the animal. The other hand holds the puncture needle and inserts it from the lower part of the right costal margin of the mouse. During the movement, to ensure that the puncture needle is at the subcutaneous site, when the needle tip reaches the right shoulder and back, push the needle plug backward to push the tumor mass into the needle tip;

　　2.6 The assistant uses ophthalmic curved forceps to clamp the underside of the tumor tissue, and at the same time, the operator quickly withdraws the puncture needle from the animal's skin;

　　2.7 The waste liquid generated during the vaccination process should be discarded together with the container in a medical waste barrel;

　　2.8 Sterilize the trocar.