Side Population Assay: A Method to Isolate Side Population of Tumor Cells from Zebrafish

- Chemotherapeutic treatment for tumors generally targets highly proliferative cells, leaving behind a small subset of the stem cell population, leading to the tumor's recapitulation. These stem cells are called the side population. Since the side population lacks specific cell markers, we use a dye efflux assay to isolate them from other tumor cells.

To begin, add Hoechst dye to the isolated zebrafish tumor cells. Hoechst enters the nucleus and binds to the A-T rich region in the minor groove of DNA. The side population of cells has a higher concentration of ABC transporters on the surface, leading to dye's rapid efflux outside the cell, displaying low fluorescence.

Take another vial of Hoesct-treated tumor cells and add verapamil to it. This vial acts as a control, because verapamil blocks the ABC transporters causing a reduction in hoechst efflux, therefore, increasing fluorescence in the side population.

Incubate both the control and sample cells in the dark. Next, counter stain cells with propidium iodide that enter the nucleus of dead cells exclusively and intercalate between DNA bases, staining the dead cells red. Finally, sort the cells using a fluorescence activated cell sorter. In the following protocol, we will isolate the side population of tumor cells from zebrafish.

- 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 4 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 FACS tube with a final volume of one milliliter.

To prepare the control, add FBS/PBS, 2.5 microliters of 100 millimolar verapamil, 15 microliters of one milligram per milliliter Hoechst dye. Add 10 to the 6 tumor cells, with a final volume of one milliliter.

Incubate the sample and the control in a 28 degrees 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, 4 degrees Celsius for six minutes. Then remove the supernatant and wash the cells with one milliliter of FBS/PBS. Spin the cells at 300 times G, 4 degrees Celsius for six minutes once more.

Once the cells are pelleted, remove the supernatant and resuspend the cells in one milliliter of FBS/PBS. Keep the samples at 4 degrees Celsius, until cell sorting. 15 minutes prior to cell sorting, add two microliters of one milligram per milliliter propidium iodide 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.