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.
1. Preparation of Cells
<ol>
	<li>Cancer cell lines
	<ol>
		<li>Culture a respective human or murine cancer cell line at 37 &#176;C in 10-30 mL of appropriate medium (e.g., RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin) and harvest the cells at sub-confluency (i.e.&#160;70-90%) using digestion with 0.05% trypsin-EDTA or some other de-attachment procedure. Determine the cell count and check for viability using a counting chamber and trypan blue staining. The fraction of viable cells should exceed 85%. 
		NOTE: Examples of human cancer cell lines harboring SP&#160;compartments include MCF7/HTB-22 and SKBR3/HTB-30 (breast cancer), A2780 and SKOV-3/HTB-77 (ovarian cancer), and A549 (lung cancer)26.</li>
	</ol>
	</li>
	<li>Ex vivo tumor tissue
	<ol>
		<li>Take a fresh surgical tumor specimen or alternatively, harvest tumor tissue from a transplantable or genetically-engineered mouse tumor model. Take 200-1,000 mg of tissue and generate a single cell suspension by mechanical disaggregation (e.g., using scissors or a scalpel) and enzymatic digestion (e.g., using a collagenase/dispase/DNase cocktail).</li>
		<li>Filter the sample through a 70 &#181;m cut-off strainer. Determine the cell count and check for viability using a counting chamber and trypan blue staining. 
		NOTE: Digestion cocktails frequently contain 200 &#181;g/mL collagenase P, 0.8 U/mL dispase and 25 &#181;g/mL DNase I, and incubation times typically range from 20 - 50 min. Ideally, tissue disaggregation is performed according to a protocol optimized for the tissue under investigation 27,28,29.</li>
	</ol>
	</li>
</ol>
2. Test Samples
<ol>
	<li>Adjust cell concentration to 1 x 106 cells/mL in fresh culture medium (a nutrient-containing vehicle is important as dye extrusion is an active energy-consuming process). Finally, transfer 1 mL of sample to an ordinary flow cytometry/FACS round-bottom tube (labelled &#39;test&#39;). 
	NOTE: For optimal results, it can be necessary that the cell concentration is titrated (for instance 5 x 105-1 x 106-2.5 x 106 cells/mL), but a lower cell concentration generally yields a higher resolution19. Only for sorting applications where a maximum number of SP cells shall be recovered, it is recommended to increase the cell concentration to up to 1 x 107 cells/mL.</li>
	<li>Add 10 &#181;M of DCV to the sample and mix well, either by gentle vortexing or resuspending for 3-5x. Proceed to inhibition controls. 
	NOTE: Optimal results might depend on titration of the triggering dye (for instance 2.5; 5; 10; 20 &#181;M), but a higher dye concentration generally yields a higher resolution19.</li>
</ol>
3. Inhibition Controls
<ol>
	<li>Transfer 250 &#181;L of the dye-containing cell suspension to a new flow cytometry/FACS round-bottom tube (labelled &#39;control&#39;). Add 50 &#181;M of verapamil or 20 &#181;M of FTC and mix well by pipetting. 
	NOTE: Verapamil and FTC are the most well-established inhibitors for SP analysis but have different efflux pump specificity: Verapamil inhibits several ABC drug transporters including ABCB1 and ABCB5, whereas FTC is specific for ABCG26,19. If the SP status of an individual sample is unknown, it is thus recommended to use both verapamil and FTC in separate tubes.</li>
</ol>
4. Staining
<ol>
	<li>Cap both the &#39;test&#39; and &#39;control&#39; tubes and incubate them at 37 &#176;C in the dark for 90 min, with gentle agitation every 15 min. Most of the dye extrusion in SP cells will occur within the first 45 min, but dye accumulation in non-SP cells (contributing to resolution) is only maximal after 75-90 min19.</li>
	<li>After staining is complete, wash the cells in 3-4 mL ice-cold PBS and resuspend the pellet in a volume of 100 &#181;L (PBS as well). Keep the cells in the dark and chilled on ice, and proceed to the staining of additional markers. Alternatively, directly perform flow cytometric analysis.</li>
</ol>
5. Costaining for Other Markers
<ol>
	<li>Add a desired panel of fluorochrome-conjugated antibodies to the DCV-stained cells, mix well and incubate the cells at 4 &#176;C in the dark for 20-30 min. Wash the cells in 3 - 4 mL ice-cold PBS and proceed to dead cell discrimination. 
	NOTE: Antibody formats that are easily compatible with DCV and virtually do not require compensation include PerCP or PerCP-Cy5.5, PE-Cy7, APC, Alexa Fluor 647, APC-Cy7, APC-H7, BV711, Alexa Fluor 750 and BV786. Formats that are compatible with DCV but may require compensation include FITC, Alexa Fluor 488, PE and BV650. Formats that are virtually not compatible with DCV include BV421, V450 and V500.</li>
	<li>Add 7-AAD (or PI) to the DCV-stained cells at the concentration recommended by the particular manufacturer and incubate the samples for 5 - 10 min in the dark. Filter the samples through 70 &#181;m cut-off strainers and proceed to flow cytometric analysis.</li>
</ol>
6. Flow Cytometric Analysis
NOTE: It is recommended to switch on the respective flow cytometric instrument during the staining process already so that everything is set as soon as the samples are ready. This includes also standard maintenance procedures such as system performance tracking and tank refilling.
<ol>
	<li>Acquire the cells on the flow cytometer and gate them on a bivariate FSC/SSC dot plot (Figure 1B). Exclude doublets and aggregates by comparing the different signals of FSC (i.e.&#160;height, width, area) (Figure 1C).
	<ol>
		<li>If a dead cell marker has been included, gate on the viable cell fraction (e.g., 7-AAD-negative) (Figure 1D). Visualize the viable singlet cells on a bivariate dot plot for &#39;blue&#39; and &#39;red&#39; DCV emission. To this end, display the &#39;blue&#39; fluorescence channel on the y-axis (e.g., 450/50) and the &#39;red&#39; fluorescence channel on the x-axis (e.g., 525/50).</li>
		<li>Switch both channels to linear mode and adjust the detector voltages correspondingly so that the SP locates to the side of the plot in the lower left quadrant. Conversely, the non-SP locates to the upper right quadrant of the plot reflecting the cell cycle distribution (G1 and G2). Gate the SP and stop the acquisition (Figure 1E). 
		NOTE: DCV-defined SP cells can be detected using flow cytometric instruments equipped with a violet laser excitation source (i.e.&#160;a 405 nm laser line). It is one specific of SP analysis that the emitted fluorescence is measured in linear mode and in two separate channels displayed on a bivariate dot plot. The &#39;blue&#39; emission of DCV is measured at approximately 450 nm (e.g., with a 450/40 standard filter) and the &#39;red&#39; emission of DCV is measured at approximately 510 - 585 nm (e.g., with a 510/50, a 525/50 or a 585/15 filter)19. Due to the particular chemistry of the assay, the separation between SP and non-SP cells is generally higher in the &#39;red&#39; fluorescence channel19.</li>
	</ol>
	</li>
	<li>Load the inhibition control(s) and acquire the data. The SP cells have now disappeared or are reduced substantially (Figure 1F). If necessary, refine the SP gate based on these data.</li>
	<li>To investigate surface marker expression by SP cells, leave the &#39;red&#39; fluorescence of DCV on the x-axis and display a respective fluorescence channel on the y-axis in logarithmic mode (e.g., APC). Adjust the detector voltages correspondingly and gate the fraction of SP cells staining positive for the investigated marker(s) (Figure 1G). Co-expression of a particular marker by SP cells yields a respective signal in the upper left quadrant of the plot30.</li>
	<li>Record the data and/or sort the SP cells.</li>
</ol>

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<li>Ho, M. M., Ng, A. V., Lam, S., Hung, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+in+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Endometrial+cancer+side-population+cells+show+prominent+migration+and+have+a+potential+to+differentiate+into+the+mesenchymal+cell+lineage%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((PTEN%2FPI3K%2FAkt+pathway+regulates+the+side+population+phenotype+and+ABCG2+activity+in+glioma+tumor+stem-like+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Cancer+stem+cells+are+enriched+in+the+side+population+cells+in+a+mouse+model+of+glioma%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Side+population+cells+have+the+characteristics+of+cancer+stem-like+cells%2Fcancer-initiating+cells+in+bone+sarcomas%5BTitle%5D)+AND+%22Br+J+Cancer%22%5BJournal%5D)">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>
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</ol>

The authors have no conflicts of interest to declare. There is also no non-author involvement in the preparation of the manuscript.

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. Mechanistically, SP discrimination harnesses active dye efflux by ABC drug transporters to detect CSCs based on low fluorescence entailing a characteristic localization at the side of the plot bottom left to the corresponding non-SP. Importantly, DCV-based SP detection is a robust and straightforward method, with the most critical steps being (i) the preparation of cells (ii) the staining process (iii) the selection of an appropriate inhibitor and (iv) the flow cytometric analysis. Because dye extrusion is an active energy-consuming process, it is particularly relevant to start off with a good cell viability; only viable (drug transporter-expressing) cells can produce a SP. Moreover, it often makes sense to optimize the ratio between dye concentration and cell density and thereby enhance the separation of SP cells (in turn, this requires titration of both DCV and cells). A general rule is that both a higher dye concentration and a lower cell concentration result in improved separation19. Key to a successful DCV experiment is the functional validation of the SP using pharmacological inhibitors of ABC drug transporter activity. As stated earlier, verapamil and FTC are among the most established inhibitors for SP analysis and have different pump specificities (FTC is specific for ABCG2, whereas verapamil inhibits several pumps). For samples with unknown SP status, it is thus recommended to use both inhibitors in separate tubes. Another option is the use of the pan-ABC drug transporter inhibitor reserpine4. However, researchers should be aware that autofluorescence of this compound can potentially interfere with the DCV signal19. Once the cells are analyzed for blue and red DCV emission, the detector voltages should be adjusted so that both populations (i.e.&#160;SP and non-SP) are visible with maximum separation. If the picture looks strange, logarithmic scaling might be the reason why (in this case, switch both channels back to linear mode).
It is also important to note that the DCV-based SP assay is neither specific for cancer nor has it been originally developed for application in oncology. Instead, it is specific for ABC drug transporters and thus also facilitates the detection and isolation of physiological (tissue) stem cells, such as lineage- Sca-1+ c-kit+ hematopoietic stem cells25. In addition, differentiated cell types establishing dedicated barriers at crucial body sites can also exhibit SP characteristics20. Applying DCV-based SP detection to primary tissues hence requires specific discrimination of the target cell population using suitable markers.
In view of the high conservation of drug efflux pumps among CSC populations9,23, DCV-based SP detection is broadly applicable, a fact that is also exemplified by its successful use across tumor entities including entities of different germ layer origin9,17,18. Other advantages include that (i) cells with direct implications in drug resistance are identified (particularly important for the tackling of clinically-relevant cancer cell subsets mediating disease recurrence) and that (ii) the detection of a functional, rather than a static, parameter might confer more assay specificity and improve the resolution19,31,32. From the conceptual/technological side, DCV-based SP detection is clearly distinct from antibody-mediated surface stainings: (i) The target structure shows intracellular localization and represents nucleic acid, not protein. (ii) The assay detects protein activity, not the mere presence or density. (iii) The target cells are defined by virtue of low fluorescence, not by a positive signal. (iv) As a DNA-binding dye, DCV is sensitive to even slight variations in the cell or dye concentration during staining19. (v) DCV fluorescence is measured in linear mode, not logarithmically. (vi) DCV fluorescence (i.e.&#160;the fluorescence generated by a single fluorophore) is measured in two separate channels, not in a single wavelength range. Despite these specifics, DCV-based SP analysis is easy to perform and evidently extends the flow cytometric toolbox for CSC research.
DCV-based SP analysis also offers many possibilities for costaining for other markers, being compatible with both conventional antibody-mediated surface stainings (numerous formats possible) and the commercial assay detecting ALDH enzymatic activity. However, we here focused on surface staining and the reader is referred to published literature4 to see methodological details on codetection of ALDH activity. In any case, DCV-triggered SP analysis should be combined with dead cell discrimination and obvious reagents for this purpose are 7-AAD and PI.
Of note, DCV-defined SP cells (and non-SP control cells) can be live-purified using the sorting application of corresponding flow cytometric instruments. Because the SP assay detects a functional parameter that only viable cells can produce, the viability of the recovered SP cells can generally be expected to be high (sometimes approximate 100%). If there should be problems with post-sort viability nonetheless, different technical parameters can be checked such as the size of the sorter nozzle, the nature of the buffering agent, and the temperature during sorting (using a 70 &#181;m nozzle we never had problems with viability, but a larger nozzle might still help for very delicate cells). The recovered cells can be subjected to downstream analyses such as qRT-PCR, Western blotting and global expression profiling (e.g., using gene array or the more recent RNASeq technology). Furthermore, isolated SP cells can be investigated for functional stem cell characteristics (e.g., single cell clonogenicity and serial transplantation), or sub-cultured to establish cell lines of enriched or even pure SP cells that might be stable for months9. In our study, we used normal medium and conventional 2D plastic flasks to sub-culture DCV-defined CSCs9, but stem cell-selective modalities such as anchorage-independent/3D culture conditions and specialized media will also be feasible. We propose that the most favorable conditions for in vitro expansion of a given SP compartment will need to be checked on an individual basis.
In summary, we here depict the detailed experimental workflow of CSC identification/isolation by means of DCV-based SP analysis. Our paper shall assist CSC researchers to implement and establish this useful method in their own labs, which should further promote the elucidation of therapeutic anti-CSC strategies for improved clinical management of cancer.

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><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 - PI)</td><td>BioLegend</td><td>420403</td><td>Ready to use</td></tr><tr><td>Standard cell culture reagents (&lt;em&gt;e.g.&lt;/em&gt;, medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, &lt;em&gt;etc.&lt;/em&gt;)</td><td>Different suppliers</td><td></td><td></td></tr><tr><td>Sample/cells of interest (&lt;em&gt;e.g.&lt;/em&gt;, 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 (FACS; 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 &amp;micro;m cut-off strainers</td><td>Different suppliers</td><td></td><td>Optional but recommended</td></tr><tr><td>Digestion enzyme mix (&lt;em&gt;e.g.&lt;/em&gt;, 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 determined using a counting chamber and trypan blue staining and 1 x 106 cells/mL (fresh medium) were stained with 10 &#181;M DCV for 90 min at 37 &#176;C in the dark. In parallel, an aliquot thereof was incubated in the presence of 20 &#181;M FTC under exactly the same conditions. Cells were washed in 4 mL PBS and an APC-conjugated antibody against ABCG2 was added to the &#39;test&#39; tube at a pre-titrated concentration. After incubation for 25 min at 4 &#176;C, cells were washed in PBS, and 7-AAD was added to label dead cells. Samples were chilled on ice and analyzed on a FACS instrument. The depicted gating strategy is more or less universally applicable to other samples even though highly heterogeneous material such as samples from tissue specimens may require pre-definition of the target cell fraction using specific surface markers (e.g., EpCAM, CD45, CD31). The results shown are intended to serve as reference for DCV-based SP detection in other labs. Hence, the relative localizations of the investigated cell populations should be roughly comparable, but surely not identical, to those obtained by other researchers with independent biological samples.
<img alt="Figure 1" src="/files/ftp_upload/55634/55634fig1.jpg" /> 
Figure 1: Workflow and Representative Results. (A) Principal flow chart for SP detection using DCV staining. Costaining for additional markers is optional, but dead cell discrimination is highly recommended. (B-G) Representative SP analysis of A2780 human ovarian cancer cells performed on a FACS instrument. Cells are gated according to FSC/SSC characteristics (B) and doublets and aggregates are excluded by comparing the different parameters of FSC (e.g., height and width) (C). Thereafter, dead cells are discriminated by gating on the 7-AAD-negative (or PI-negative) cell fraction (D). Live single cells are then displayed on a linear bivariate dot plot for blue and red DCV emission and the SP (and the corresponding non-SP) is gated (E). The same analysis is now done with a respective inhibition control (in this case FTC was used), and this should lead to almost-complete disappearance of cells within the SP gate (F). Finally, the identified SP can be more closely characterized, e.g., by determining the ABC drug transporter(s) that might be responsible for dye extrusion hence conferring the SP phenotype (in this case ABCG2, in line with FTC sensitivity). (G). FTC, fumitremorgin C; ALDH, aldehyde dehydrogenase. <a href="http://cloudflare.jove.com/files/ftp_upload/55634/55634fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

harnessing-dna-dye-triggered-side-population-phenotype-to-detect

Nanyang Technological University

Research

JoVE Journal

Cancer Research

7.6K Views.  Kantonsspital St. Gallen. 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.

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

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Alternative Laser Excitation Method for Easy Side Population Cell Detection

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Author Spotlight: Innovative Laser Techniques for Hoechst Staining to Analyze Side Population Cells

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Immunology and Infection

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Cancer Side Population Discrimination Assay: A Method to Isolate Cancer Stem-like Cells Using Hoechst Dye Efflux Assay

Source: Gilormini, M. et al. <a href="https://www.jove.com/video/53958" target="_blank">Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics.</a> J. Vis. Exp. (2016)
In this video, we describe the isolation of a subpopulation of cancer stem-like cells from head and neck squamous carcinoma cell lines using fluorescence-activated cell sorter or FACS. The isolated side population of cells can be used for further downstream analysis.

Test dyskryminacji populacji po stronie raka: metoda izolowania rakowych komórek macierzystych za pomocą testu wypływu barwnika Hoechsta

Source: Gilormini, M. et al. Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics. J. ...