The overall goal of this procedure is to demonstrate a method for the production of metastatic tumors in black six mice. This method can help answer key questions in the field of immunotherapy like how investigative agents affect the immune response in the lungs. The main advantage of this technique is that it's experiment metastases.
So the results are reliable and relatively consistent. To begin, place cultures of B16-BL6 melanoma cells, which should be at approximately 70%confluency, in a sterile hood. Next, add one milliliter of 05%trypsin EDTA to each dish and leave the cells for one minute.
After aspirating this solution, proceed to add one milliliter of trypsin to every plate, and then return the cells to the incubator. After 10 minutes, move the cells to a sterile hood and add four milliliters of serum free RPMI medium to each plate. Then collect the cells with a sterile motorized pipette.
To break up clumps that could potentially clog the ejection needle, press the tip of the pipette against the bottom of the plate and expel the cells. After repeating this process several times, recollect the cells and transfer them to a 15 milliter conical tube. Then remove a sample of the cell suspension, introduce it into a hemocytometer, and determine the cell density.
EqualLy allocate the cell suspension into four 15 milliliter tubes. Then centrifuge the tubes, and afterwards decant the supernatant. Proceed to label each of the tubes with a different cell density-0, 0.063, 0.125, 0.25, or 0.5 million cells per milliliter.
Then add an appropriate volume of serum free RPMI to each conical to produce these final concentrations. Confirm the desired cell densities using a hemocytometer. Next, allocate 500 microliters of each suspension into labeled 1.8 milliliter tubes placed on ice.
After all B16-BL6 cell suspensions have been prepared, select a mouse. Holding it by the tail, guide the animal rear first into a restrainer. When the mouse's torso is in the main chamber and its tail outside of the apparatus, proceed to pull the animal towards the end of the restrainer.
Next, insert the restrainer plunger and continue to push on the plunger until the mouse is secure. Once the animal is in place, rotate the mouse 90 degrees such that one of the lateral tail veins is facing upwards. Then use an alcohol pad to vigorously clean the side of the mouse's tail where the vein is most visible.
To prepare for injection, first collect 300 microliters of the 0.5 million cells per milliliter suspension into a syringe. Afterwards, eject bubbles from the syringe by inverting it, flicking its side, and pushing the plunger. Once bubbles have been removed, eject the cell culture to yield a final volume of 250 microliters in the syringe.
Proceed to extend the mouse tail with the non-dominant hand. Hold it so that forefinger elevates the proximal end of the tail and the thumb depresses the distal portion. With the dominant hand, insert the needle into the vein towards the distal end of the tail at a minute downward angle.
Then insert the needle further, adjusting it so that it matches the angle of the tail vein. After the needle has been inserted approximately one centimeter into the tail vein, begin to push the plunger while holding the syringe steady. Stop any bleeding by holding gauze against the entry site.
Once the cell suspension has been ejected, remove the needle from the vein and discard it. Next, remove the plunger from the restrainer and place the mouse in a recipient cage. This protocol aimed to identify the optimal number of B16-BL6 cells to inject to create a useful model of metastatic melanoma.
Shown here are experimental foci indicated by arrows formed in lungs two to three weeks after the injection of five different cell densities. When 500, 000 cells per milliliter were injected, foci completely covered the lungs. In the example shown here, 102 foci were counted.
In contrast, lungs were only sparsely covered in foci when 62, 500 cells per milliliter or 125, 000 cells per milliliter were injected. Respectively, these densities resulted in lung foci counts of one and 17. Using this method, it was determined that the optimal concentration of cells to inject was 250, 000 cells per milliliter.
This density resulted in lungs with about 65 foci, a number at which organs were neither completely covered nor too sparsely covered by foci. Further enumeration of these data when graphed demonstrates an approximately linear relationship between lung foci and cell culture density above 125, 000 cells per milliliter as shown here. Once mastered, this technique can be performed in under an hour, number of mice depending, if performed properly.
While attempting procedure, it's important to remember to try and inject an equivalent number of cells per mouse. Following this procedure, other methods can be performed, such as drug delivery, to answer questions like how potential therapies affect a number of resulting foci. After its development, this technique paved the way for cancer researchers to explore the constituents of metastatic cancer in melanoma in black six mice.
After watching this video, you should have a good understanding of how to collect and prepare B16-BL6 cells, restrain mice, prepare needles, and inject an equivalent number of cells into each tail vein. Don't forget that working with mammalian culture and needles can be extremely hazardous, and precautions, such as avoiding needle sticks and wearing protective gear should always be taken.
