The overall goal of the following experiment is to test the metastatic abilities of human cancer cells by tail vein injections. In immunodeficient mice, the cells are prepared for injection. The cells are then injected into immunodeficient mice, which will generate lung metastases after one to two months.
The mice are dissected and lung metastases are quantified and cultured. Results of this type of assay indicate the extent of metastases of a particular type of cancer cell. Hi, I am Sonali Mohanty from Dr.LE's Lab in the Department of Biomedical Genetics at the University of Rochester.
Today we will show you a procedure to perform experimental metastasis assay. We use this procedure in our lab to study cancer metastasis. So let's get started.
Begin with cells grown to about 70%Co fluency aspirate media from the plate and gently wash once with one XPBS. Next, add two milliliters of 0.05%Trypsin inversing. Gently rock the plate to facilitate cell detachment.
Observe the cells under a microscope as they detach. Add an ample volume of media containing FBS to quench the trips in activity. Then transfer to a 50 milliliter tube.
Count the cells using a hemo cytometer. Once the concentration has been determined, centrifuge at about 200 times gravity for three minutes without disturbing the cell pellet. Carefully remove the media Resus suspending an appropriate volume of Hank's balance buffer solution or HBSS to reach a final concentration of about five times 10 to the six cells per milliliter.
Next, filter through a Falcon's 70 micrometer cell strainer. To exclude large cell aggregates, place the tip of the pipette directly onto the filter, placed over a five milliliter culture tube. Quickly eject the suspension through the filter into the tube.
Count the cells again using trian blue to determine viability. The viability should be greater than 90%prior to injection. Then dilute to a final concentration of 2.5 times 10 to the six cells per milliliter.
Keep on ice. Gently grab the tail of an immunodeficient mouse and pull it into the mouse restrainer with its back against the slit and its tail sticking outta the small opening in the back. Slowly slide the ring inward along the slit.
Once the ring catches the mouth of the mouse, lock it into place. The mouse should not be able to move freely, but should have a normal rate of breathing. Find the major tail veins.
Four major blood vessels are present in a mouse tail. The arteries are located on the dorsal and ventral sides of the tail. The veins are located on the lateral sides.
Draw at least 200 microliters of the prepared cells into a one milliliter syringe. Attach a 30 gauge half needle and push out any air bubbles. The final volume in the syringe should be 200 microliters.
Prepare to inject into the distal end of the tail. So if the first trial fails, a more proximal region of the tail could be used for a second try. After wiping the injection site with 70%ethanol, pull the tail straight.
Hold the tip with the thumb and support the point for injection with the index finger. Insert the needle into the vein and inject the cells. Make sure the needle and syringe are parallel to the vein during injection.
Otherwise, the needle will poke through the vessel wall and cells will be injected into the adjacent tail tissues. Withdraw the needle if the procedure went successfully. Blood should perfuse from the injection site.
Press a clean cotton ball to facilitate flossing and palpate the tail upwards to push any residual sample into circulation. Release the mouse from the restrainer and return it to the cage. Record the number of injection trials and the number of cells injected in a laboratory notebook.
Determine the viability of cells after injection. As before this step provides reassurance that cells stay alive throughout the injection process. At the end of an injection experiment, the viability of cells will decline, but should remain above 80%As an optional step, spin down the leftover cells and rinse the pellets with PBS once.
Then freeze the pellets at minus 80 degrees Celsius for future analyses such as western blotting. To confirm gene expression on knockdowns After one to two months, dissect the mice to determine the locations of metastases. Begin by placing the euthanized mouse on a dissection station.
Spray the mouse generously with ethanol. Cut open the rib cage to expose the lungs and heart. Then carefully excise them and place them in PBS.
The lung is the primary site for metastasis since it contains the first capillary bed that cells encounter. After they enter the circulation, remove the heart and rinse the lungs in PBS then observe each lobe of the lung under a dissecting microscope. Count the number of detectable metastases on both sides of the lung, then at the numbers of lung metastases on all four lobes together to determine the total number of lung metastases.
Next, isolate individual lung metastases by excision Using scissors. Transfer each metastasis to a 70 micrometer cell strainer and mince it using the end of a sterile needle cap. Rinse the cell strainer with several milliliters of medium and collect the cells that pass through the filter.
In a culture dish, each dish contain the cells from one metastasis. Incubate the cells at 37 degrees Celsius for at least four days without disturbance. Initially, both cancer cells and fibroblast cells will grow on the plates, but gradually the fibroblast cells will die out and be replaced by cancer cells.
If the cancer cells carry any drug resistant genes, select the cells with corresponding antibiotics After three days, wash away any blood or tissue debris, debris on the dish with PBS and add fresh medium. Assess the purity of the derived cells by immuno staining with antibodies against human specific proteins here and anti-human menting antibody is used even in the absence of drug selection. Greater than 99%of the cells should be human.
The newly derived cells can now be injected again into immunodeficient mice to test their metastatic abilities at the end of this assay. A highly metastatic cancer cell line typically gives rise to many lung metastases, while very few lung metastases will come from a poorly metastatic cancer cell line. These images show lung metastases two months after injection of the human melanoma cell line.
A 3 7 5 sm. The cells derived using this method usually give rise to more metastasis than the parental line when they're tested. Again using this assay, We have just shown you the procedure to perform experimental metastasis assay.
When doing this procedure, it is very important to remember to ensure that the cells are injected into the tail vein, but not the its and tail tissue. So that's it. Thank you for watching and good luck with your experiments.
