Orthotopic Injection into the Mammary Fat Pad: Establishing Breast Cancer in Mice

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 d