Encyclopedia of Experiments: Cancer Research
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Transcript
- T-cell acute lymphoblastic leukemia, or T-ALL, is a type of cancer that occurs when lymphocytes fail to mature and remain as immature cells called lymphoblasts. These abnormal cells are ineffective in fighting infection. As these immature cells divide rapidly, they outnumber healthy blood cells and can travel to the organs such as the spleen, liver, and CNS.
Tumors in zebrafish are similar to those formed in humans. Therefore, zebrafish is the preferred model to generate T-ALL tumor cells.
Begin by selecting the transformed fishes that exhibit fluorescent signals, indicating the spread of tumor cells. Remove the euthanized fish's head and wash the body cavity with heparin solution to remove excess red blood cells. Now, inject a suitable buffer into the body cavity. Apply gentle pressure from outside and collect the buffer containing tumor cells in a microfuge tube.
Stain the cells with trypan blue and observe them under a microscope. Live tumor cells appear fluorescent and colorless when stained with trypan blue, as the negatively charged cell membrane excludes the negative trypan blue from entering inside the cell. Trypan blue enters dead cells through the porous membrane, staining them blue. In the following protocol, we will isolate GFP-labeled T-ALL tumor cells from adult zebrafish.
- Prepare 20 milliliters of 10% heat-inactivated FBS in 0.9X PBS. Add 1 milliliter of the FBS/PBS solution to a vial of a heparin sodium salt containing 300 units of the anticoagulant. Then, using an epifluorescent microscope, select fish in which tumor cells have invaded 50% of the body or fish that are identified as having difficulty eating or swimming.
Euthanize the fish by transferring it to a solution of 1 to 2 milligrams per milliliter tricaine in fish water. Observe the animal for gill movement and heartbeat to confirm euthanasia.
After removing the head of the fish, inject with a pipette, 100 microliters of heparin solution into its body cavity to remove excess red blood cells. Aspirate all liquid that pours out and pipette it into a 1.5 millimeter microcentrifuge tube. Then, using fresh pipette tips, wash the body cavity two more times or until the washing solution does not contain any blood and discard the liquid collected from all washes.
To collect leukemia cells, inject 100 microliters of FBS-PBS solution into the prewashed body cavity. Under a dissecting microscope, apply gentle pressure to the body of the fish with a pipette tip to force out the tumor cells. Collect the released liquid into a 1.5 milliliter microcentrifuge tube. Repeat isolation until most of the tumor cells are harvested.
Collect all cells in the same 1.5 milliliter microcentrifuge tube and keep the obtained suspension at room temperature. Then mix the isolated tumor cells by gently pipetting to dissociate cell clumps. Filter the cell suspension through a 40 micrometer mesh filter and wash the filter one to two times with 100 microliters of FBS-PBS.
Once filtered, keep the cells at room temperature. Finally, in a new tube, mix 2 microliters of the cell suspension with 18 microliters of 0.04% sterile trypan blue solution. Load 10 microliters of the obtained suspension onto a hemocytometer and count the viable fluorescent tumor cells.
- It is important to collect a clean sample of T-ALL cells. The concentration of Hoechst 33342 used here is based on the number of live tumor cells collected. Additional cellular debris or non-tumor cells may interfere with the assay.
