This video protocol demonstrates the identification and isolation of slow dividing cells in human glioblastoma using carboxy fluorescein SDI eter, CFSE. Glioblastoma tumor cells are first grown using the neuros sphere assay. Then the resulting spheres are dissociated into single cells and stained with CFSE.
The CFSE loaded cells are plated again. In neuros sphere culture, the resulting spheres are dissociated into single cells and run through the flow cytometry machine. Slow and fast dividing cells are identified and sorted based on fluorescence intensity as a function of cell division.
Frequency Heterogeneity is an essential feature in cancer. This heterogeneity supports tumor robustness and presents a great obstacle to the development of fully effective treatment. Therefore, it is critical to develop tools and assays to study and understand this heterogeneity that is defined genetically phenotypically and functionally.
In this video protocol, we describe a standing method using the flues and I-C-F-S-C. This technique allows the identification and separation by flow cytometry of individual subpopulation within a culture of human glioblastoma derived cells possessing different functional characteristics. We have used this technique to isolate and characterize a tumor population of cells exhibiting a slow dividing rate, which is a functional property that has been shown to be associated with tumor initiation Ins cancer Gliomas sphere Culture is established and maintained using the neuros sphere assay, which is described in a separate video at the address on the screen.
When the gliomas spheres reach a size of 150 to 200 microns in diameter, the medium containing the spheres is removed and placed in an appropriate sized sterile tissue culture tube and centrifuged at 800 RPM or 110 G for five minutes at room temperature. Then the supernatant is discarded and the pelle of spheres is resuspended in one mil of pre-war tripsin EDTA and incubated at 37 degrees in a water bath for three to five minutes. An equal volume of trypsin inhibitors then added with gentle pipetting up and down to dissociate the spheres.
The cell suspension is again centrifuge. The SUP natin is removed and the cells are resuspended in one mil of neuros stem cell. Basal medium, 10 Microliters of the single cell suspension is mixed well with 90 microliters of triam blue and a cell count is performed For every 1 million cells to be stained.
One mil of staining reagent is prepared by adding one microliter of five millimolar CFSE dye to one mil of neuro stem cell basal medium to give a final staining concentration of five micromolar. Then the staining solution is mixed properly. The desired number of cells are then transferred to a separate tube centrifuged and the supra natin is removed.
The CFSE staining solution is then added to the pellet and gently mixed until homogeneity and incubated at room temperature for 10 minutes to stop the staining process. Five to 10 volumes of ice cold neuro stem cell basal medium is added to the CFSE loaded cells and mixed Well. The cell suspension Is then centrifuged and the supra natin is discarded.
At this point. There should appear a elle of cells at the bottom of the tube with a green tint indicating the staining was successful. To remove excess CFSE dye two to three washes are performed by resus, suspending the pellet in one mil of neuro stem cell basal medium centrifuging and discarding the supinate.
After the third wash, the cells are resuspended in one mil of neuro stem cell basal medium, and a cell count is performed as described previously to plate the cells. Complete neuros stem cell medium is supplemented with EGF at a final concentration of 20 nanograms per ml. Then the cells from the CFSE loaded and unloaded groups are added to their respective tubes to reach a final concentration of 50, 000 cells per ml.
Then the cell suspensions are mixed thoroughly And plated in sterile tissue culture flasks. The flasks are then labeled. The cells can be monitored under a fluorescence microscope to verify CFSE staining and then placed in a 37 degree 5%CO2 humidified incubator for five to 10 days.
To verify CFSE staining using flow cytometry, 500, 000 cells from CFSE loaded and unloaded negative control groups are transferred to separate fax tubes. The flow cytometer is adjusted based on its user manual Instructions for related parameters. First, the unloaded control cells are run and the events are recorded based on forward and side scatter properties, and then in a histogram plot based on cell frequency versus CFSE intensity, the main cell population is then gated to verify the level of CFSE fluorescence intensity.
Next, the CFSE loaded cells are analyzed using the same parameters used for control cells, as is evident. The CFSE positive cells exhibit fluorescence intensity that is orders of magnitude higher than control cells. When the glioma spheres have reached an average size of approximately 150 to 200 microns in diameter.
The spheres are passaged and a single cell suspension is prepared from both CFSE loaded and unloaded groups as described earlier. Then the cells are resuspended in an appropriate volume of sterile one times PBS and a cell count is performed to exclude dead or damaged cells. When sorting by flow cytometry, propidium iodide is added at a concentration of one microliter per ml of cell suspension to collect slow dividing and fast dividing cells.
When sorting two sterile 15 mil tissue culture tubes are prepared each containing one mil of complete neuro stem cell medium. Then the cells are placed on ice while transferring to the flow cytometry facility. First, the cell suspension from the unloaded cell group is run through the flow cytometer.
The events are plotted based on forward scatter versus side scatter to exclude dead or damaged cells. Cells are plotted based on side scatter versus PI reactivity and a single live cell population is gated as population one. Then a homogenous cell population from population one is selected based on forward and side scattered properties and gated as population Two And population two is then plotted in a histogram based on cell frequency versus CFSE intensity set on a logarithmic scale Using the same Parameters and gating strategy.
The CFSE labeled cells are run through the flow cytometer and analyzed. Subsequently, the bottom 85%of cells are gated as fast dividing and the top 5%are gated as slow dividing Cells. These different cell Populations of interest are selected and sorted into separate 15 mil sterile tissue culture tubes containing one mil of complete neuro stem cell medium.
The sorting threshold rate should be adjusted not to exceed 2, 500 events per second. After finishing the Sort, the cells are centrifuged sup natin is discarded and the pellet is resuspended in an appropriate volume of medium and a cell count is Performed. Then the cells from The different sorted populations are plated at a density of 50, 000 cells per ml.
In complete neuro stem cell medium, supplemented with 20 nanograms per ML of EGF cells are then incubated for five to 10 Days to grow spheres. This Is an example of cells. Soon after loading with CFSE dye, all the cells present an intense green fluorescence as visualized under the fluorescence microscope.
This is an image of a representative glioma sphere derived from A-C-F-S-E loaded cell six days after plating. Notice that some of the cells still have very bright CFSE staining. This is an example of a flow cytometry histogram that shows that CFSE intensity decays over time.
Blue represents the cells freshly loaded with CFSE showing fluorescence intensity. That is orders of magnitude higher than unloaded cells represented in red. Green represents the fluorescence intensity having decayed over a six day period post loading with CFSE due to the cells dividing.
These are representative spheres grown seven days after isolating fast and slow dividing populations. Sphere forming frequencies are not different between the two groups, but the CFSC retaining cells grow slower and result in smaller spheres as compared to CFSC diluting cells. Using CFSC to identify and isolate subpopulation of cells based on the rate of cell division is a starting point for downstream experiment and for further characterizations.
This protocol provided an example to identify slow dividing cells in human glioma derived culture, but it also can be applied to other tumor cell type or to non-cancerous systems. Thank you for watching and we hope you found this protocol helpful for your research.
