Analysis of side population cells is one of the commonly used methods to isolate and identify cancer stem cells from tumor tissues and tumor cell lines. This assay is convenient, fast, and cost-effective. This method can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on stemness properties of tumor cells.
Many factors can influence the percentage of SP cells in this analysis, so it is critical to explore the proper conditions before the formal experiment. Seed tumor cells in a six-well plate, and incubate them in a 37-degree Celsius incubator supplied with 5%carbon dioxide. When the cell density reaches about 90%aspirate the culture medium, and wash the cells twice with three milliliters of PBS.
Then, add 500 microliters of 0.25%trypsin-EDTA to each well, and incubate the plate at 37 degrees Celsius for one to three minutes. Gently tap the plate to detach the cells, add three milliliters of PBS supplemented with 2%FBS to each well, and pipette the cells up and down three to five times to disperse the cell clumps. Examine the cell suspension under the microscope.
If cell clumps are present, pass the suspension through a 70-micrometer strainer. Transfer the cells to a 15-milliliter centrifuge tube, and centrifuge them at 200 times g for five minutes. Remove the supernatant, and resuspend the cells in three milliliters of PBS supplemented with 2%FBS, pipetting the cells up and down three to five times to mix.
Count cells under the microscope using a hemocytometer, and dilute them in PBS supplemented with 2%FPS to a final concentration of one times 10 to the six cells per milliliter. Prepare two five-milliliter, polystyrene, round-bottom test tubes, and add one milliliter of the cell suspension to each tube. Label one tube as a test tube and the other as a blocker control tube.
Prepare a solution of blocker by diluting it to the appropriate concentration. Add it to the blocker control tube and mix well, then incubate the tube at 37 degrees Celsius for 30 minutes. Shake the tube every 10 minutes.
Prepare a solution of Hoechst 33342 by diluting it to the appropriate concentration. Add it to the test tube and blocker control tube separately and mix well, then incubate the tubes at 37 degrees Celsius for 60 minutes. Shake the tubes every 10 minutes.
After the incubation, centrifuge the cells for five minutes at 200 times g and four degrees Celsius, then carefully aspirate the supernatant. Resuspend the cells in each tube with one milliliter of ice-cold PBS supplemented with 2%FBS, then pipette the cells up and down to mix. Add one microliter of one-milligram-per-milliliter propidium iodide, or PI, to each tube and mix.
Click the Parameters tab in the flow cytometer software. First, choose the parameters of FSC, SSC, Hoechst Blue, Hoechst Red, and PI, respectively. Second, choose the logarithmic scale for the PI parameter.
Finally, choose area and width scales for the FSC, SSC, Hoechst Blue, Hoechst Red, and PI parameters, respectively. Run the cells from the test tube first, then run the cells from the blocker control tube using the same voltages and gates. Collect 20 to 100, 000 events from each sample to analyze the percentage of SP cells.
This method was used to detect the proportion of SP cells in the MDA-MB-231 human breast cancer cell line. The dot plot of FSC-A versus PI-A showed a population of dead cells, cell debris, and the main population of PI negative cells, which was subjected to further analysis. A single cell population gated from the dot plot of FSC-A versus FSC-W and the dot plot of SSC-A versus SSC-W was used to analyze the proportion of SP cells, which was about 0.9%in untreated cells and about 0.09%in cells treated with reserpine.
Different staining concentrations of Hoechst 33342 were tested on MDA-MB-435 cells, and it was determined that two micrograms per milliliter was optimal for SP analysis. Low concentrations made it difficult to distinguish SP cells from other populations, while high concentrations caused SP cells to disappear. Verapamil and reserpine were tested to determine the proper blocker for the MDA-MB-435 cells.
About 0.4%of cells expelled the dye after verapamil treatment, but the ratio dropped to about 0.1%after reserpine treatment, demonstrating that reserpine is a more appropriate blocker for this cell line. This protocol was also used to determine the proportion of SP cells in A549 human lung adenocarcinoma cells treated with STAT3 activator colivelin and in T47D human breast cancer cells treated with FRA1 inhibitor SKLB816. This assay provides clues to the regulatory effect of signaling pathways on the features of cancer stem cells and facilitates the discovery of new mechanisms, which can ultimately guide the targeted therapy of tumors.
