First, establish the desired breast cancer cell line in culture. The day before the operation, shave the mouse's abdomen and use depilatory cream to remove the fur and expose the inguinal nipples. On the day of the operation, collect the cells, anesthetize the mouse and confirm a lack of response to toe pinch. Using tweezers, pinch the skin around the nipple to create an uplifted tunnel.
Then, with the syringe of collected cells, pierce the skin. The needle should follow the tunnel until the tip comes close to the nipple. Gently move the needle tip up into the mammary fat pad, just below the nipple. Remove the tweezers to form a skin tent with the needle and then slowly inject the cells.
Lastly, retract the syringe, taking care not to disturb the round bump surrounding the nipple. Injected cells and subsequent tumors express luciferase and can be tracked using bioluminescent imaging. In the example protocol, we will show the establishment of murine orthotopic breast cancer. On the day before the operation, manually restrain the recipient mouse and turn the animal belly side up to the fur to be shaved around the fourth nipples.
Wipe the fur with a thin layer of depilatory cream. Used distilled water to remove the cream after 30 to 60 seconds. And dry the mouse with soft paper towels. On the day of the operation, dilute the murine breast cancer cell suspension to a 2 x 10 to the 5th cells per milliliter of PBS concentration in a sterile microcentrifuge tube on ice.
Before each injection, load one, one milliliter syringe equipped with a .45 by 16 needle, with 50 microliters of cells per mouse. And confirm a lack of response to toe pinch in each sedated animal. Apply ointment to the animals eyes, and use tweezers to tent the skin around the fourth nipple of the first mouse to create a tunnel for the syringe to follow.
Hold the syringe with the bevel facing upwards and subcutaneously enter the skin at five to 10 milliliters from the nipple, following the tunnel until the needle tip almost reaches the nipple. - Using tweezers to tent the skin around the first nipple creates a tunnel for the syringe to follow. The needle should be entered between the tweezer tips and should ride up, instead of depressing into the tissue. Carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple.
And release the skin tent to allow a slow injection of the cancer cells. - Upon insertion, carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple, and that you feel resistance to ensure delivery of the cells to the correct location. When all of the cells have been injected, turn the needle slightly to the side and retract the syringe slowly. Use a cotton swab to apply pressure to the needle wound for 10 to 20 seconds, to make sure there is no fluid seepage.
The injected nipple should appear as a transparent white flat sphere, with the nipple in the center