This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.
Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2.
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.
1. Preparing Glioblastoma Single Cell Suspension
2. Staining with Cell Trace Carboxyfluorescein Succinimidyl Ester (CFSE) Green Fluorescent Dye
3. CFSE Loading Verification
4. Isolation of the Slow-dividing [i.e. CFSE Retaining] Cell Population Using Fluorescent Activated Cell Sorting (FACS)
5. Representative Results
After CFSE loading, all the cells present an intense green fluorescence as visualized under the microscope (Figure 1). This green-tinted color can be seen even with the naked eye (see the video). Analyzing these cells with flow cytometry also shows highly intense green fluorescence compared to control cells (Figure 2). Upon plating CFSE loaded cells in neurosphere culture, these cells proliferate and form 150-200 μm gliomaspheres in 5-10 days. As the cells proliferate, the fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. Glioblastomas are functionally heterogeneous and are composed of cells proliferating on different time scales. The method described in this protocol enables one to identify and isolate fast and slow dividing cells by virtue of their ability to dilute or retain CFSE, respectively (Figure 3, also see the video).
Flow cytometry analysis of dissociated gliomaspheres generated from CFSE-loaded cells in comparison to unloaded cells demonstrates diverse CFSE retention properties (Figure 4). Fast dividing cells (bottom 85%) or slow dividing cells (the top 5% – CFSEhigh) exhibit gliomapshere forming ability when cultured in the neurosphere assay conditions (Figure 5).
Figure 1. Dissociated tumor cells immediately after loading with CFSE dye. The cells exhibit bright green color demonstrating successful labeling. Original magnification; 10 x.
Figure 2. CFSE loading confirmation using flow cytometry. A) Unloaded cells, B) CFSE loaded cells and C) Comparing CFSE fluorescence intensity in loaded versus unloaded cells. Note that there is several orders of magnitude difference in the fluorescence intensity between the labeled and unlabeled cells.
Figure 3. A representative gliomasphere derived from a CFSE-loaded cell 6 days after plating. Some cells exhibit very bright CFSE staining. Original magnification; 63 x.
Figure 4. Representative flow cytometry plots/histograms for isolation of fast and slow dividing cells 6 days after CFSE staining. A) FSC vs. SSC to show the entire cell population, B) SSC vs. PI reactivity to exclude dead or damaged cells, C) FSC vs. SSC to gate a homogenous live cell population, D) Demonstrates the presence of CFSE retaining cells 6 days after CFSE labeling as evidenced by a greater fluorescence intensity compared to the negative control. The histogram also shows that the CFSE intensity decayed over time. E) Histogram displaying the gating strategy to identify CFSE retaining cells (top 5%) and CFSE diluting cells (bottom 85%).
Figure 5. Representative gliomaspheres generated from A) fast-dividing cells (CFSElow) and B) slow-dividing cells (CFSEhigh). Original magnification: 10x and 20x respectively. C) Representative cell count obtained from fast or slow dividing cell-derived culture at passage time. P<0.05, t-test. Click here to view larger figure.
An increasing number of studies have demonstrated the importance of a slow-dividing sub-compartment of cells in cancer which contribute to tumor formation and treatment resistance2-8. Therefore it is critical to describe and standardize experimental methods enabling the study of this specific subpopulation of cells or more generally to compare subpopulations with different growth rates. Using CFSE to identify and isolate sub-fractions of cells based on their rate of cell division is a starting point to further characterizing these specific cellular fractions, helping to advance our understanding of the heterogeneity described in tumors. In this protocol we gave the example of identifying and isolating a slow-dividing population within glioblastoma, but this protocol can also be applied to other tumor cells, including and not limited to breast, pancreatic and skin cancers. Downstream analysis of these populations can be performed and can include functional studies comparing proliferative potential2, tumor formation ability2, treatment sensitivity, and (epi)genetic comparisons. This methodology can also be used with non-cancerous systems and can be applied, for example, to somatic stem/precursor cells.
Critical technical points within the procedure involved adequate dissociation of cells with trypsin, and sufficient resuspension of cells in the staining media to establish a proper density and ensure homogenous staining. A small sample of freshly stained cells should be checked by flow cytometry to ensure homogenous labeling characterized by a Gaussian-like narrow profile (see Figure 2B). In the case of heterogeneous initial labeling, a pre-sort can be performed in order to improve peak resolution with narrower range of CFSE fluorescence. However it should be ensured that this pre-sort does not select for specific sizes. The use of propidium iodide (PI) labeling is important for identifying live cells and for dead/damaged population removal from the working gate, due to the bias that dead cells can introduce in downstream applications. It is worth noting the particularly high fluorescence intensity of the CFSE labeling and its potential to bleed into other channels when using neighboring fluorochromes in the spectrum, such as the fluorochrome phycoerythrin (PE). It is crucial to note that multiplex labeling experiments with CFSE and fluorochrome-conjugated antibodies may require a compensation process at the acquisition or analysis time. Be sure to have all the necessary controls that include unstained cells (providing the level of autofluorescence), singly stained cells and your specific combination. Multiple-color labeling in combination with CFSE works best with fluorochromes such as Pacific Blue or Allophycocyanin (APC). If the purpose of the experiment is to quantify the absolute number of cell divisions over a defined period of time, the CFSE Mean Fluorescence Intensity (MFI) has to be measured at a minimum of two different time points2 with the first one at least 24 hours post CFSE loading. CFSE intensity drops drastically within the first 24 hours in the absence of cell division but then stabilizes and decreases in relation to cell division. It is also necessary to include a control with non-proliferative CFSE loaded cells providing the baseline for CFSE intensity decay not related to cell division.
The authors have nothing to disclose.
This work was supported by the Florida Center for Brain Tumor Research, the Preston A. Wells Jr. Center for Brain Tumor Therapy and the National Institutes of Health (1R21CA141020-01).
Name of the reagent | Type | Company | Catalogue number |
NeuroCult NSC Basal Medium (Human) | Medium | Stem Cell Technologies | 05750 |
NeuroCult NSC Proliferation Supplements (Human) | Medium supplement | Stem Cell Technologies | 05753 |
%0.05 trypsin-EDTA | Reagent | Gibco | 25300-062 |
Soybean trypsin inhibitor | Reagent | Sigma | T6522 |
Penicillin/Streptomycin | Reagent | Gibco | 15140-122 |
T25 flask | Culture ware | Nalge Nunc international | 136196 |
T80 flask | Culture ware | Nalge Nunc international | 178905 |
15 ml tubes | Culture ware | BD Falcon | 352096 |
50 ml tubes | Culture ware | BD Falcon | 352070 |
EGF | Growth factor | R&D | 2028-EG |
CellTrace CFSE Cell Proliferation Kit | Fluorescent Cell Division Tracking Dye | Molecular Probes, Invitrogen | C34554 |