Start by adding trypsin into a Petri dish containing labeled breast cancer cells to detach them from the surface. Add medium containing serum to stop the action of trypsin. Transfer the cell suspension to a conical tube and centrifuge it to pellet cell.
Discard the supernatant and resuspend the cells in phosphate-buffered saline. Place the tube on ice to slow down cell metabolism. Load this cell suspension containing the required amount of cells in a Luer lock syringe and fix a needle onto it.
Place a mouse in a rodent restrainer with its tail outside. Dip the tail in warm water to dilate the veins. There are two veins on the lateral sides and an artery on the ventral side of the tail. Wipe the tail with alcohol, insert the needle, and inject the cell suspension.
Once in the bloodstream, the injected cancer cells invade into organs, such as the lungs, and proliferate. Slowly remove the needle and use a sterile gauze on the injection site to apply pressure to stop bleeding. Return the mouse to its cage and ensure a full recovery. In the following protocol, we demonstrate the injection of labeled metastatic cancer cells into a mouse model to quantify breast cancer metastasis and colonization.
Aspirate the media and rinse the cell plates with 1X PBS. Trypsinize the cells with 5 milliliters of trypsin per 15-centimeter plate for two to five minutes and 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. Add the wash to the same conical tube. Count the cells using an automated cell counter to determine the total cell number.
Next, centrifuge the cells at 122 times G for three minutes and aspirate the supernatant. Resuspend the cells in 1X PBS at the desired concentration. Here, 25,000 cells are injected into each mouse in 100 microliters of PBS, so the resuspended cells are at 250,000 cells per milliliter. Keep the cell suspensions on ice until injection.
Working in a hood at the animal facility, gently, but thoroughly mix the cells by inverting the tube or using a 1-milliliter syringe to ensure that they are uniformly resuspended. Now, load a 1-milliliter Luer lock syringe with cell suspension and expel excess air bubbles. Place a 1/2-inch, 30-gauge needle on the syringe with the bevel up and expel air bubbles.
Gently place the mouse in a rodent restrainer. The lateral tail vein 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. Use an alcohol wipe to clean the tail. Then insert the needle into the tail vein, bevel side up, and inject 100 microliters of cell suspension. 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. Slowly remove the needle, and, using a sterile gauze, apply pressure to the injection site to stop any bleeding. Return the mouse to its cage and monitor for 15 minutes to ensure full recovery.
