The overall goal of this procedure is to isolate side population cells from a zebrafish model of myc-induced T-cell acute lymmphoblastic leukemia. The side population assay has been used to identify stem and progenitor cell populations in many normal and cancer tissues. But it hasn't yet been applied to any zebrafish cancer models.
The main advantage of this technique is that it allows the prospective isolation of a population of cells in zebrafish T-ALL that may be enriched for cancer stem cell activity. Although the method we describe here has broad applications in zebrafish leukemia research it may also be applicable to other malignant and non-malignant zebrafish cell types. Individuals new to this method may struggle because the method is sensitive to subtle changes.
Even though the method seems straightforward, optimization is required for each cell type tested. Prepare 20 milliliters of 10 percent heat-inactivated FBS in 0.9x PBS. Add one 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 percent of the body, or fish that are identified as having difficulty eating or swimming. Euthanize the fish by transferring it to a solution of one to two 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 milliliter 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 pre-washed 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 two microliters of the cell suspension with 18 microliters of 0.04 percent 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 three three three four two used here is based on the number of live tumor cells collected. Additional cellular debris or non-tumor cells may interfere with the assay. Prior to cell staining, dilute Hoechst dye in a nuclease-free water to obtain one milligram per milliliter working solution.
Store the prepared solution covered with aluminum foil at four degrees Celsius. Then, dissolve 49.1 milligrams of verapamil in one milliliter of DMSO to obtain a 100 micromolar inhibitor solution. Next, prepare the sample by adding FBS/PBS, 2.5 microliters of DMSO, 15 microliters of one milligram per milliliter Hoechst dye, and 10 to the six tumor cells to a fax tube with a final volume of one milliliter.
To prepare the control, by adding FBS/PBS, 2.5 microliters of 100 million molar verapamil, 15 microliters of one milligram per milliliter Hoechst dye, add 10 to the six tumor cells with a final volume of one milliliter. Incubate the sample and the control in a 28 degree Celsius water bath in the dark for 120 minutes. After the incubation, place the cells immediately on ice and keep them in the dark.
Centrifuge the cells at 300 times G four degrees Celsius for six minutes. Then, remove the supernatint and wash the cells with one milliliter of FBS/PBS. Spin the cells at 300 times G four degrees Celsius for six minutes once more.
Once the cells are pelleted remove the supernatint and re-suspend the cells in one milliliter of FBS/PBS. Keep the samples at four degrees Celsius until cell sorting. 15 minutes prior to cell sorting, add two microliters of one milligram per milliliter propidium iodine stock solution per each one milliliter of the cell suspension.
Then, vortex the samples well. Analyze the stained zebrafish T-ALL cell suspensions using a fluorescence activated cell sorter. Excite samples with a UV laser for Hoechst dye analysis and a 488 nanometer laser for the detection of propidium iodide or GFP positive T-ALL cells.
The number of cells, concentration of dye, incubation temperature, and incubation length may need to be optimized for each different cell type used. And changes to each of these conditions can drastically alter the results of the assay. To identify the side population among T-ALL cells isolated from zebrafish, first cellular debris and cell clumps are excluded.
Propidium iodide intake and GFP expression in a single cell population are then analyzed to identify live tumor cells. To visualize the side population, blue and red emissions of the Hoechst dye are then compared. The side population appears as a dim tail of cells extending from the left side of the main cell population towards the blue fluorescence axis.
Presented here are results of the side population analysis carried out for two different tumor cell samples. A side population was identified in both specimens. However, different tumor samples contained varied numbers of the side population cells.
Regardless of the tumor sample analyzed, treatment of the sample with an efflux inhibitor of verapamil resulted in the disappearance of the signal observed for the previously identified cells further confirming the side population phenotype. Once mastered, the side population technique on a zebrafish T-ALL can be done in less than four hours if it is performed properly and as we presented here. While attempting this procedure, it's important to remember the steps that have been optimized for zebrafish T-ALL such as incubation temperature and time, dye concentration, and efflux inhibitor.
These may need to be changed for different experimental systems. Following this procedure, other methods, like RNA isolation, or in vivo transplantation assays, can be performed with sorted side population cells to address questions regarding the functionality and the gene regulation of the side population cells. After watching this video, you should have a good understanding of how to isolate the side population of cells from zebrafish T-cell acute lymphoblastic leukemia.
