The overall goal of this procedure is to detect putative cancer stem cell populations in biological samples based on dye extrusion capacity of the side population cells. This method addresses a key question in cancer stem cell research regarding reliable means for detection and purification of cancer stem cells from biological samples. The main advantage of this technique is that it deploys functional markers and therefore providing better resolution.
In addition, expression of these ABC drug transporters is highly conserved among cancer stem cells and therefore we can use this technique for many different cancer entities. The implications of this technique extends toward the discovery of anti-cancer stem cell therapeutics because it facilitates downstream analysis such as gene expression profiling and target validation in highly purified cancer cells. So this method can provide insight into the characteristics of cancer stem cells.
We can also use this method to characterize as a systems comprising ABC drug transporter expressing populations such as the bone marrow. Demonstrating the procedure will be Elisabeth Hoflehner, a technician from my laboratory. To begin, culture the desired human cancer cell line at 37 degrees celsius in 10 to 30 milliliters of RPMI 1640 medium supplemented with 10%FBS, two millimeter L-glutamine, and antibiotics.
When cells reach approximately 70 to 90%confluence, remove the medium and add two milliliters of 0.05%trypsin-EDTA to detach the cells from the culture dish surface. Then, wash the cells with 10 milliliters of culture medium, transfer the suspension to a clean 15 milliliter tube, and spin it at 250 times G at four degrees celsius for five minutes. Once the cells are pelleted, discard the suprenatent and resuspend the pellet in one milliliter of culture medium.
Mix a small aliquot of the cell suspension with trypan blue solution and count the cells using a hemocytometer. To prepare test samples, suspend the cells in fresh culture medium to obtain a cell suspension of one time ten to the six cells per milliliter. After adjusting the cell density, transfer three milliliters of the sample to a 15 milliliter round bottom tube labeled as test.
Then, add 10 micromolar of violet dye to the sample and gently vortex the suspension. Next, prepare inhibition controls by transferring 500 microliters of the dye containing cell suspension to two flow cytometry tubes labeled as control. Add 50 micromolar of Verapamil to the first.
And 20 micromolar of Fumitremorgin C to the second tube. And mix the solutions by gently pipetting. Cover all tubes with caps and incubate them at 37 degrees celsius in the dark for 90 minutes.
Gently agitate the samples every 15 minutes. Once the staining is completed, wash the inhibition controls in three to four milliliters of ice cold PBS. And prepare aliquots of the test sample for subsequent costaining with antibodies.
Wash this aliquots as well and filter all samples through 70 micron cut off strainers to remove potential cell aggregates. And then centrifuge the suspensions at 250 times G at four degrees celsius for five minutes. After resuspending the resulting pellet in 100 microliters of PBS, maintain the cells on ice protected from light.
Next, add an appropriate panel of flurochrome conjugated antibodies to the cells prestained with violet dye. Vortex the suspensions and incubate them at four degrees celsius in the dark for 20 to 30 minutes. After incubation, wash the cells in three to four milliliters of ice cold PBS, filter the sample if desired.
And then centrifuge at 250 times G at four degrees celsius for five minutes. Discard the supernatent and resuspend the pelleted cells in 100 microliters of PBS. Finally, add 7-AAD to the cells prestained with violet dye.
And incubate the samples for five to 10 minutes in the dark. To analyze the cells using the flow cytometer, gate an appropriate cell population on a FSC SSC dot plot. Then, using different FSC signals, exclude from the selected population all cell aggregates.
Exclude cells revealing positive 7-AAD staining to obtain the population of viable cells. To create a dot plot for blue and red dye emission, display the blue fluorescent channel on the y-axis and the red fluorescent channel on the x-axis. Switch both channels to the linear mode and adjust the detector voltage accordingly so that the side population is located in the lower left part of the plot.
Then, gate the side population. And stop the acquisition. Next, load the inhibition controls and acquire the data.
Finally, create a dot plot displaying the red florescence of violet dye on the x-axis and an appropriate florescence channel on the y-axis plotted in logarithmic mode. Load the sample then adjust the detector voltages accordingly and identify the faction of the side population cells that express the investigated surface. Then, record the data.
Presented here are representative results of the side population analysis carried out in human ovarian cancer cells. The side population distinguished by the analysis of blue and red-violet emission was identified in the left lower quadrant of the dot plot and constituted 0.70%of the whole analyzed population. The later use of efflux inhibitors resulted in the disappearance of the florescence signal observed for the previously identified cells confirming their side population phenotype.
Finally, the human ovarian cells were also characterized in terms of ABCG2 expression. ABCG2 positive staining was observed in 0.67%of cells corresponding to the identified side population. Once mastered, this procedure can be done in approximately two to three hours if performed properly.
When attempting this procedure, it is important to remember that the side population phenotype strictly depends on an optimal dye concentration to cell density ratio. Hydration of both variables is therefore highly recommended prior to the experiment. Following this procedure, other methods like gene expression profiling can be performed in order to further characterize side population cells hence to identify novel cancer stem cell specific targets for future drug development.
After its development, this technique paved the way for researchers in the field of stem cell biology to explore the characteristics of tissue stem cells and cancer stem cells, both in mice and men. After watching this video, you should have a good understanding on how to perform violet dye triggered side population SA and how to detect putative cancer stem cells in biological samples in your own lab. Don't forget that working with biological samples can be extremely hazardous and that precautions such as the use of PPE and biological safety cabinets should be taken when working with this procedure.
