Maximilian Boesch is supported by an Erwin Schr&#246;dinger Fellowship of the Austrian Science Fund FWF (grant number J-3807).

This protocol is in full compliance with standard Institutional Ethical Review Board guidelines. If human or animal tissue is investigated using the protocol depicted here, researchers are obliged to have approval from the relevant review board of their institution or country.
Caution: Standard precautions for the safe handling of biological samples apply to this protocol. This includes wearing gloves and a lab coat, and performing the work under a biosafety cabinet whenever possible.
<p c...

<ol>
	<li>Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+cancer+stem+cells+-+what+challenges+do+they+pose?.">Tackling the cancer stem cells - what challenges do they pose?.</a> Nat Rev Drug Discov. 13 (7), 497-512 (2014).</li><li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+Cancer+Stem+Cells:+Rationale+for+Targeting+the+Stem+Cell+Niche.">Heterogeneity of Cancer Stem Cells: Rationale for Targeting the Stem Cell Niche.</a> Biochim Biophys Acta. 1866 (2), 276-289 (2016).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+cancer+relapse+and+metastasis+by+inhibiting+cancer+stemness.">Suppression of cancer relapse and metastasis by inhibiting cancer stemness.</a> Proc Natl Acad Sci U S A. 112 (6), 1839-1844 (2015).</li><li>Greve, B., Kelsch, R., Spaniol, K., Eich, H. T., G&#246;tte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+in+cancer+stem+cell+analysis+and+separation.">Flow cytometry in cancer stem cell analysis and separation.</a> Cytometry A. 81 (4), 284-293 (2012).</li><li>Marcato, P., Dean, C. A., Giacomantonio, C. A., Lee, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase:+its+role+as+a+cancer+stem+cell+marker+comes+down+to+the+specific+isoform.">Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.</a> Cell Cycle. 10 (9), 1378-1384 (2011).</li><li>Golebiewska, A., Brons, N. H., Bjerkvig, R., Niclou, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+side+population+assay+in+stem+cell+and+cancer+stem+cell+research.">Critical appraisal of the side population assay in stem cell and cancer stem cell research.</a> Cell Stem Cell. 8 (2), 136-147 (2011).</li><li>Hirschmann-Jax, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+&amp;#34;side+population&amp;#34;+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells.">A distinct &amp;#34;side population&amp;#34; of cells with high drug efflux capacity in human tumor cells.</a> Proc Natl Acad Sci U S A. 101 (39), 14228-14233 (2004).</li><li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nat Med. 7 (9), 1028-1034 (2001).</li><li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+side+population+of+ovarian+cancer+cells+defines+a+heterogeneous+compartment+exhibiting+stem+cell+characteristics.">The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.</a> Oncotarget. 5 (16), 7027-7039 (2014).</li><li>Szotek, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+side+population+defines+cells+with+stem+cell-like+characteristics+and+Mullerian+Inhibiting+Substance+responsiveness.">Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.</a> Proc Natl Acad Sci U S A. 103 (30), 11154-11159 (2006).</li><li>Brown, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+benign+and+malignant+prostate+epithelial+Hoechst+33342+side+populations.">Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.</a> Prostate. 67 (13), 1384-1396 (2007).</li><li>Mathew, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2-mediated+DyeCycle+Violet+efflux+defined+side+population+in+benign+and+malignant+prostate.">ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate.</a> Cell Cycle. 8 (7), 1053-1061 (2009).</li><li>Engelmann, K., Shen, H., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF7+side+population+cells+with+characteristics+of+cancer+stem/progenitor+cells+express+the+tumor+antigen+MUC1.">MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1.</a> Cancer Res. 68 (7), 2419-2426 (2008).</li><li>Ho, M. M., Ng, A. V., Lam, S., Hung, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells.">Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.</a> Cancer Res. 67 (10), 4827-4833 (2007).</li><li>Kato, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+cancer+side-population+cells+show+prominent+migration+and+have+a+potential+to+differentiate+into+the+mesenchymal+cell+lineage.">Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage.</a> Am J Pathol. 176 (1), 381-392 (2010).</li><li>Bleau, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN/PI3K/Akt+pathway+regulates+the+side+population+phenotype+and+ABCG2+activity+in+glioma+tumor+stem-like+cells.">PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.</a> Cell Stem Cell. 4 (3), 226-235 (2009).</li><li>Harris, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+are+enriched+in+the+side+population+cells+in+a+mouse+model+of+glioma.">Cancer stem cells are enriched in the side population cells in a mouse model of glioma.</a> Cancer Res. 68 (24), 10051-10059 (2008).</li><li>Murase, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+cells+have+the+characteristics+of+cancer+stem-like+cells/cancer-initiating+cells+in+bone+sarcomas.">Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.</a> Br J Cancer. 101 (8), 1425-1432 (2009).</li><li>Boesch, M., Wolf, D., Sopper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Stem+Cell+Detection+Using+the+DyeCycle-Triggered+Side+Population+Phenotype.">Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype.</a> Stem Cells Int. 2016, 1652389 (2016).</li><li>Golebiewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+human+glioblastoma+is+non-tumorigenic+and+characterizes+brain+endothelial+cells.">Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.</a> Brain. 136 (PT 5), 1462-1475 (2013).</li><li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transporter-mediated+protection+of+cancer+stem+cells+from+ionophore+antibiotics.">Drug transporter-mediated protection of cancer stem cells from ionophore antibiotics.</a> Stem Cells Transl Med. 4 (9), 1028-1032 (2015).</li><li>Fukunaga-Kalabis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenascin-C+promotes+melanoma+progression+by+maintaining+the+ABCB5-positive+side+population.">Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.</a> Oncogene. 29 (46), 6115-6124 (2010).</li><li>Dean, M., Fojo, T., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+stem+cells+and+drug+resistance.">Tumour stem cells and drug resistance.</a> Nat Rev Cancer. 5 (4), 275-284 (2005).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183 (4), 1797-1806 (1996).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+side+population+in+human+cancer+cell+lines.">High prevalence of side population in human cancer cell lines.</a> Oncoscience. 3 (3-4), 85-87 (2016).</li><li>Ali, M. Y., Anand, S. V., Tangella, K., Ramkumar, D., Saif, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Primary+Human+Colon+Tumor+Cells+from+Surgical+Tissues+and+Culturing+Them+Directly+on+Soft+Elastic+Substrates+for+Traction+Cytometry.">Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J Vis Exp. (100), e52532 (2015).</li><li>Azari, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+human+glioblastoma+multiforme+tumor+cells+using+the+neurosphere+assay.">Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay.</a> J Vis Exp. (56), e3633 (2011).</li><li>Hasselbach, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+high+grade+glioma+cell+culture+from+surgical+specimens+for+use+in+clinically+relevant+animal+models+and+3D+immunochemistry.">Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.</a> J Vis Exp. (83), e51088 (2014).</li><li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DyeCycle+Violet+used+for+side+population+detection+is+a+substrate+of+P-glycoprotein.">DyeCycle Violet used for side population detection is a substrate of P-glycoprotein.</a> Cytometry A. 81 (6), 517-522 (2012).</li><li>Patrawala, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+is+enriched+in+tumorigenic,+stem-like+cancer+cells,+whereas+ABCG2++and+ABCG2-+cancer+cells+are+similarly+tumorigenic.">Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic.</a> Cancer Res. 65 (14), 6207-6219 (2005).</li><li>Guha, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+promotes+apoptosis+resistance+of+breast+cancer+cells+and+enrichment+of+side+population+cells+with+cancer+stem+cell-like+properties+via+a+signaling+cascade+involving+galectin-3,+alpha9+nicotinic+acetylcholine+receptor+and+STAT3.">Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, alpha9 nicotinic acetylcholine receptor and STAT3.</a> Breast Cancer Res Treat. 145 (1), 5-22 (2014).</li></ol>

Progress in the clinical management of cancer also depends on the development of treatment modalities that target CSCs. Methods allowing the reliable isolation of CSCs from biological samples will accelerate the identification of suitable targets and are therefore of critical importance in this endeavor. SP analysis is an established and valuable technique to identify CSC populations that express ABC drug transporters and the implementation of DCV has extended its applicability to standard flow cytometric instruments. Me...

The efficacy of primary cancer therapies has improved substantially over the last decade due to compelling advances in the genetic and molecular understanding of the disease and the advent of targeted drugs such as therapeutic antibodies and small molecule inhibitors. In contrast, metastatic and recurrent disease is still typically incurable, and morbidity and mortality remain high in these clinical settings. CSCs&#160;represent a distinct subpopulation within tumors and are endowed with canonical stem cell properties such as clonogenicity/tumorigenicity, multi-drug resistance and asymmetric cell division1,2. Thus, CSCs not only drive metastatic progression and tumor heterogeneity, but also persist during treatment to predispose the patient to relapse. Therapeutic CSC elimination is therefore an important medical need to prevent disease recurrence and allow long-term cure of cancer3.
Identification of vulnerabilities and elucidation of strategies to eradicate CSCs heavily depends on methods that allow their purification from biological samples for subsequent expression profiling and/or functional investigation. In turn, such methods rely on surface, intracellular or functional markers that are specific for these cells. CSC-specific surface markers include, but are not limited to, CD44, CD133, CD24 and CD90, and have been used to identify CSC populations in a variety of tumor entities including breast cancer and colon cancer4. Another marker, aldehyde dehydrogenase (ALDH), shows intracellular localization and can be functionally detected providing a respective substrate whose enzymatic conversion produces light. Using this test, CSC populations have been identified in diverse tumor entities as well5. A complementary method, commonly referred to as SP&#160;analysis and portrayed here in methodological detail, harnesses active dye efflux by ABC drug transporters to identify small populations of fluorescence-dim stem-like cells6,7,8. To achieve this, a given sample is incubated in the presence of a lipophilic DNA-binding fluorophore which enters all cells through passive diffusion and targets nuclear and mitochondrial DNA for binding. Non-CSCs devoid of ABC drug transporter expression accumulate the dye resulting in bright fluorescence, whereas CSCs actively extrude the dye which reduces fluorescence. Pharmacological inhibition of drug transporter activity abrogates and functionally confirms the SP phenotype and should be used for control purpose. CSC populations exhibiting SP characteristics have been disclosed, amongst others, in ovarian cancer9,10, prostate cancer11,12, breast cancer13, lung cancer14, endometrial cancer15, glioma16,17 and bone sarcoma18. Importantly, the SP assay is compatible with both cancer cell lines and primary tumor tissue, even though primary material poses additional challenges such as the requirement for a specific tumor cell discrimination strategy (certain host cell populations can exhibit SP characteristics as well)19,20.
The two most established SP-conferring drug transporters are ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD3388,9,21; however, other drug transporters can be a molecular determinant of the SP phenotype too (e.g., ABCB5)22. ABCB1 can be efficiently blocked with verapamil whereas the activity of ABCG2 can be specifically abrogated with fumitremorgin C (FTC)6,19. A particular strength of SP analysis is that it can be combined with other stainings (e.g., surface markers and ALDH) and that it allows live cell recovery thus being compatible with downstream functional investigations. Moreover, SP detection is broadly applicable owing to the high conservation of ABC drug transporters among CSC populations9,23.
Originally, SP detection has been performed using Hoechst 33342 as a triggering dye24. This dye achieves excellent resolution but requires ultraviolet laser excitation; hence its applicability is naturally limited to high-end flow cytometric instruments. The advent of DyeCycle Violet (DCV)25 has opened new avenues for SP analysis and extended the applicability of this method to standard flow cytometric instruments lacking an ultraviolet laser source (a violet laser source suffices to resolve DCV-SP cells). Importantly, Hoechst 33342 and DCV share common pump specificities, indicating that either dye should identify the same cell populations.
Here, we provide a detailed experimental protocol of DCV-based SP analysis for quick and easy reproduction in independent labs. We thus perceive our article as a resource for CSC researchers that should contribute to the optimization and standardization of this useful cell-biological method.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vybrant DyeCycle Violet</td>
			<td>Thermo Fisher Scientific</td>
			<td>V35003</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Fumitremorgin C</td>
			<td>Sigma-Aldrich</td>
			<td>F9054</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr>
			<td>7-AAD (or propidium iodide)</td>
			<td>BioLegend</td>
			<td>420403</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Standard cell culture reagents (e.g., medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, etc.)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample/cells of interest (e.g., human or murine cancer cell line, human or murine tumor tissue)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometric instrument (analyzer or cell sorter)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Violet laser source required</td>
		</tr>
		<tr>
			<td>FACS tubes (round-bottom)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Tubes with cap recommended</td>
		</tr>
		<tr>
			<td>70 &#181;m cut-off strainers</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional but recommended</td>
		</tr>
		<tr>
			<td>Digestion enzyme mix (e.g., collagenase/dispase/Dnase)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Only relevant for tissue dissociation</td>
		</tr>
		<tr>
			<td>Flurochrome-conjugated antibodies against surface markers of interest</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Kit</td>
			<td>STEMCELL Technologies</td>
			<td>01700</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Provided is a representative SP analysis of A2780 cells, a human ovarian cancer cell line previously shown to harbor a SP accounting for less than 1% of cells9. Cells were cultured at 37 &#176;C in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin and harvested at 85% confluency using treatment with 0.05% trypsin-EDTA. Cells were washed in PBS and filtered through 70 &#181;m cut-off strainers. The cell count was det...

Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

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.

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Research

JoVE Journal

Cancer Research

7.6K visualizações.  Kantonsspital St. Gallen. 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 AB...