Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells
Transcrição
To prepare the Renca tumor cells, first, attach the cell culture with 0.25% trypsin in HBSS after rinsing with PBS. Neutralize the reaction after 5 minutes with complete RPMI medium. Transfer the cells to a conical tube for centrifugation and resuspend the pellet in fresh HBSS.
After counting, adjust the volume to a 2 times 10 to the sixth cells per milliliter concentration in HBSS, and draw the cells into a 1-milliliter syringe equipped with an 18-gauge needle. Next, place a sterile surgical drape over a heating pad and confirm the appropriate level of sedation by toe pinch. Remove the hair from the left flank of the mouse and apply vet ointment to the eyes.
Then, swab the implantation site with 5% povidone-iodine antiseptic, and use a sterile scalpel to make a single 1 to 1.5-centimeter incision on the left flank, taking care not to penetrate the peritoneum. Using scissors, separate the dermis from the peritoneum, dabbing the incision site with sterile cotton gauze to remove any blood. Then, grasp the head with the left hand and support the flank with the right hand to locate the spleen through the peritoneum.
Using one finger from the right hand, palpate the mouse from underneath. The kidneys should become visible. Keeping a firm hold on the animal to provide tension across the peritoneum and kidney, have an assistant slowly deliver 0.1 milliliters of the Renca cells through the intact peritoneum into the center of the kidney. Hold the needle in place for 5 to 10 seconds to reduce the backflow of the cells. Then, close the incision with a thin layer of cyanoacrylate tissue adhesive.
