Tail Vein Injection: A Method to Administer Cancer Cells for Metastatic Studies in a Mouse Model

1. Tail vein injection of labeled cancer cells

NOTE: Step 1.2.4 has been optimized for 4T1 cells growing in syngeneic BALB/c mice. If other cancer cell lines and mouse strains are used, the number of cells injected, and the length of the assay should first be optimized.

1. Expand the 4T1 cell lines generated in step 2 on two 15 cm dishes in complete growth media so that excess cells are available on the day of injection.
2. Prepare the cells for tail vein injection as follows:
   1. Aspirate the media and rinse the cell plates with 1x PBS.
   2. Trypsinize the cells with 5 mL of trypsin per 15 cm plate for 2-5 minutes (cells should freely rinse off the bottom of the well). Transfer all of the cells to a conical tube. Wash remaining cells from the tissue culture dish with enough complete growth media to quench the trypsin and add the wash to the same conical tube.
   3. Count the cells using an automated cell counter to determine the total cell number.
   4. Centrifuge the cells at 122 x g for 3 minutes, aspirate the supernatant, and resuspend the cells in 1x PBS at the desired concentration. Here, 2.5 x 10⁴ cells are injected into each mouse in 100 μL of PBS, so the resuspend cells at 2.5 x 10⁵ cells/mL. Keep the cell suspensions on ice until injection.
      NOTE: it is important to limit the amount of time between trypsinization of the cells and the tail vein injection to roughly 1 hour.
3. Inject 4T1 cells from step 1.2.5 into mice via the lateral tail vein as follows:
   1. Working in a hood at the animal facility, gently but thoroughly mix the cells by inverting the tube or using a 1 mL syringe to ensure that they are uniformly resuspended. Always ensure the cells are resuspended prior to loading the syringe.
   2. Load a 1 mL Luer-lock syringe with cell suspension and expel excess air bubbles. Place a ½ inch, 30-gauge needle on the syringe with the bevel up and expel air bubbles.
   3. Gently place the mouse in a rodent restrainer.
   4. The lateral tail veins should be visible and dilated. If not, gently pinch the base of the tail and dip the tail in warm tap water to dilate the veins.
      NOTE: Dilation of the veins may also be achieved by placing the mouse cage under a heating lamp and/or on top of a heating pad.
   5. Use an alcohol wipe to clean the tail. Insert the needle into the tail vein, bevel side up, and inject 100 μL of cell suspension.
      NOTE: If the needle is inserted properly into the vein, it should easily slide slightly forward and back, and there should not be resistance when the plunger is pushed. Successful injections should also result in a "flush" in which the blue color of the vein turns white for a few seconds following the injection.
4. Slowly remove the needle and using a sterile gauze, apply pressure to the injection site to stop any bleeding.
5. Return the mouse to its cage and monitor for 15 minutes to ensure full recovery. Mice should be checked for signs of pain or distress 3x weekly.
6. Monitor mice for metastasis formation and growth for 3-6 weeks (cell line and mouse strain dependent) using an in vivo live animal imaging device (see steps 1.5 and 1.6).