Encyclopedia of Experiments: Cancer Research
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Transcript
For IV injection, concentrate cells to 0.5 to 1 million cells per milliliter using ice-cold 1x PBS to dilute or resuspend the cells. Expect that approximately 1 milliliter of cell suspension will be needed for every 10 embryos. To assemble the injection apparatus, mount a needle onto the syringe and then extend the syringe needle with a 3 to 5-centimeter long piece of tubing.
Break the tip of the borosilicate needle using fine forceps. Load the syringe with 50 to 200 microliters of the cancer cell suspension and insert the borosilicate needle into the tubing. Inspect the needle for cell clogging and air bubbles. If cell clogging is observed within the capillary, remove the capillary, resuspend the cells, and replace it with a new capillary.
Use the plunger to push out any bubbles. Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface, which is normally only slightly wider than the diameter of the borosilicate needle tip and located midway between the embryo and the weighing dish wall.
It is generally easier to inject at the point immediately adjacent to the vein bifurcation. Press the tip of the needle against the blood vessel wall and apply gentle pressure in the same direction as the blood flow. If necessary, use a cotton swab to help anchor or stabilize the vessel that is being injected. Gently depress the syringe plunger for 2 to 10 seconds until the desired volume is injected.
Discard the embryo if excessive bleeding or clear liquid accumulation appears at the injection site. Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells. Cover the injected embryo in the weighing dish with the lid and return it to the incubator. Repeat the procedure until all embryos are injected.
