This work was funded by the Natural Science Foundation of China 81572599, 81773124, and 81972787; Natural Science Foundation of Tianjin City (China) 19JCYBJC27300; Tianjin People&#8217;s Hospital &#38;&#160;Nankai University Collaborative Research Grant 2016rmnk005; Fundamental Research Funds for the Central Universities, Nankai University 63191153.

1. Cell preparation
<ol>
	<li>Cell digestion and neutralization
	<ol>
		<li>Seed tumor cells (such as MDA-MB-231 cells) in a 6 well plate, and culture them in a 37 &#176;C incubator supplied with 5% CO2.</li>
		<li>Harvest cells when their density reaches about 90%. Aspirate the culture medium thoroughly and wash the cells 2x with 3 mL of phosphate buffered saline (PBS). 
		NOTE: To examine the effects of signaling pathway inhibitors (e.g., FRA1 inhibitor), or activators (e.g., STAT3 activator) on stemn...

<ol>
	<li>Reya, T., Morrison, S. J., Clarke, M. F., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+cancer,+and+cancer+stem+cells.">Stem cells, cancer, and cancer stem cells.</a> Nature. 414 (6859), 105-111 (2001).</li><li>Batlle, E., Clevers, H. <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+revisited.">Cancer stem cells revisited.</a> Nature Medicine. 23 (10), 1124-1134 (2017).</li><li>Eramo, 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=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death &amp; Differentiation. 15 (3), 504-514 (2008).</li><li>Kahlert, U. 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=CD133/CD15+defines+distinct+cell+subpopulations+with+differential+in+vitro+clonogenic+activity+and+stem+cell-related+gene+expression+profile+in+in+vitro+propagated+glioblastoma+multiforme-derived+cell+line+with+a+PNET-like+component.">CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component.</a> Folia Neuropathologica. 50 (4), 357-368 (2012).</li><li>Bailey, J. 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=DCLK1+marks+a+morphologically+distinct+subpopulation+of+cells+with+stem+cell+properties+in+preinvasive+pancreatic+cancer.">DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer.</a> Gastroenterology. 146 (1), 245-256 (2014).</li><li>Hurt, E. M., Kawasaki, B. T., Klarmann, G. J., Thomas, S. B., Farrar, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44++CD24(-)+prostate+cells+are+early+cancer+progenitor/stem+cells+that+provide+a+model+for+patients+with+poor+prognosis.">CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis.</a> British Journal of Cancer. 98 (4), 756-765 (2008).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proceedings of the National Academy of Science of the United States of America. 100 (7), 3983-3988 (2003).</li><li>Yang, Z. F., 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=Significance+of+CD90++cancer+stem+cells+in+human+liver+cancer.">Significance of CD90+ cancer stem cells in human liver cancer.</a> Cancer Cell. 13 (2), 153-166 (2008).</li><li>Schulenburg, 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+in+basic+science+and+in+translational+oncology:+can+we+translate+into+clinical+application.">Cancer stem cells in basic science and in translational oncology: can we translate into clinical application.</a> Journal of Hematology &amp; Oncolcology. 8, 16 (2015).</li><li>Park, J. W., Park, J. M., Park, D. M., Kim, D. Y., Kim, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells+Antigen-1+Enriches+for+a+Cancer+Stem+Cell-Like+Subpopulation+in+Mouse+Gastric+Cancer.">Stem Cells Antigen-1 Enriches for a Cancer Stem Cell-Like Subpopulation in Mouse Gastric Cancer.</a> Stem Cells. 34 (5), 1177-1187 (2016).</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> Journal of Experimental Medicine. 183 (4), 1797-1806 (1996).</li><li>Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+identification+and+sorting+using+the+Hoechst+33342+side+population+(SP).">Stem cell identification and sorting using the Hoechst 33342 side population (SP).</a> Current Protocols in Cytometry. , 18 (2005).</li><li>Begicevic, R. R., Falasca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABC+Transporters+in+Cancer+Stem+Cells:+Beyond+Chemoresistance.">ABC Transporters in Cancer Stem Cells: Beyond Chemoresistance.</a> International Journal of Molecular Sciences. 18 (11), 2362 (2017).</li><li>Rabindran, S. K., Ross, D. D., Doyle, L. A., Yang, W., Greenberger, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fumitremorgin+C+reverses+multidrug+resistance+in+cells+transfected+with+the+breast+cancer+resistance+protein.">Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein.</a> Cancer Research. 60 (1), 47-50 (2000).</li><li>Takara, 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=Differential+effects+of+calcium+antagonists+on+ABCG2/BCRP-mediated+drug+resistance+and+transport+in+SN-38-resistant+HeLa+cells.">Differential effects of calcium antagonists on ABCG2/BCRP-mediated drug resistance and transport in SN-38-resistant HeLa cells.</a> Molecular Medicine Reports. 5 (3), 603-609 (2012).</li><li>Matsson, P., Pedersen, J. M., Norinder, U., Bergstrom, C. A., Artursson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+specific+and+general+inhibitors+of+the+three+major+human+ATP-binding+cassette+transporters+P-gp,+BCRP+and+MRP2+among+registered+drugs.">Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs.</a> Pharmaceutical Research. 26 (8), 1816-1831 (2009).</li><li>Challen, G. A., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+side+order+of+stem+cells:+the+SP+phenotype.">A side order of stem cells: the SP phenotype.</a> Stem Cells. 24 (1), 3-12 (2006).</li><li>Wu, C., Alman, B. A. <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+in+human+cancers.">Side population cells in human cancers.</a> Cancer Letters. 268 (1), 1-9 (2008).</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 Research. 65 (14), 6207-6219 (2005).</li><li>Yamada, 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=Nasal+Colivelin+treatment+ameliorates+memory+impairment+related+to+Alzheimer's+disease.">Nasal Colivelin treatment ameliorates memory impairment related to Alzheimer's disease.</a> Neuropsychopharmacology. 33 (8), 2020-2032 (2008).</li><li>Marotta, L. 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=The+JAK2/STAT3+signaling+pathway+is+required+for+growth+of+CD44(+)CD24(-)+stem+cell-like+breast+cancer+cells+in+human+tumors.">The JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(-) stem cell-like breast cancer cells in human tumors.</a> Journal of Clinical Investigation. 121 (7), 2723-2735 (2011).</li><li>Zhang, C. 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=From+Lead+to+Drug+Candidate:+Optimization+of+3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine+Derivatives+as+Agents+for+the+Treatment+of+Triple+Negative+Breast+Cancer.">From Lead to Drug Candidate: Optimization of 3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine Derivatives as Agents for the Treatment of Triple Negative Breast Cancer.</a> Journal of Medicnal Chemistry. 59 (21), 9788-9805 (2016).</li><li>Tam, W. 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=Protein+kinase+C+alpha+is+a+central+signaling+node+and+therapeutic+target+for+breast+cancer+stem+cells.">Protein kinase C alpha is a central signaling node and therapeutic target for breast cancer stem cells.</a> Cancer Cell. 24 (3), 347-364 (2013).</li><li>Ibrahim, S. F., Diercks, A. H., Petersen, T. W., van den Engh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analyses+as+a+critical+parameter+in+defining+the+side+population+(SP)+phenotype.">Kinetic analyses as a critical parameter in defining the side population (SP) phenotype.</a> Experimental Cell Research. 313 (9), 1921-1926 (2007).</li><li>Petriz, J. <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+of+the+side+population+(SP).">Flow cytometry of the side population (SP).</a> Current Protocol in Cytometry. , 23 (2013).</li><li>Hiraga, T., Ito, S., Nakamura, H. <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+MDA-MB-231+human+breast+cancer+cells+exhibits+cancer+stem+cell-like+properties+without+higher+bone-metastatic+potential.">Side population in MDA-MB-231 human breast cancer cells exhibits cancer stem cell-like properties without higher bone-metastatic potential.</a> Oncology Reports. 25 (1), 289-296 (2011).</li><li>Shen, W., 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=ICAM3+mediates+inflammatory+signaling+to+promote+cancer+cell+stemness.">ICAM3 mediates inflammatory signaling to promote cancer cell stemness.</a> Cancer Letters. 422, 29-43 (2018).</li><li>Koh, S. Y., Moon, J. Y., Unno, T., Cho, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baicalein+Suppresses+Stem+Cell-Like+Characteristics+in+Radio-+and+Chemoresistant+MDA-MB-231+Human+Breast+Cancer+Cells+through+Up-Regulation+of+IFIT2.">Baicalein Suppresses Stem Cell-Like Characteristics in Radio- and Chemoresistant MDA-MB-231 Human Breast Cancer Cells through Up-Regulation of IFIT2.</a> Nutrients. 11 (3), 624 (2019).</li><li>Lee, H., Park, S., Kim, J. B., Kim, J., Kim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrapped+doxorubicin+nanoparticles+for+the+treatment+of+metastatic+anoikis-resistant+cancer+cells.">Entrapped doxorubicin nanoparticles for the treatment of metastatic anoikis-resistant cancer cells.</a> Cancer Letters. 332 (1), 110-119 (2013).</li><li>Ota, 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=ADAM23+is+downregulated+in+side+population+and+suppresses+lung+metastasis+of+lung+carcinoma+cells.">ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells.</a> Cancer Science. 107 (4), 433-443 (2016).</li><li>Wei, 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=The+mechanisms+for+lung+cancer+risk+of+PM2.5+:+Induction+of+epithelial-mesenchymal+transition+and+cancer+stem+cell+properties+in+human+non-small+cell+lung+cancer+cells.">The mechanisms for lung cancer risk of PM2.5 : Induction of epithelial-mesenchymal transition and cancer stem cell properties in human non-small cell lung cancer cells.</a> Environmental Toxicology. 32 (11), 2341-2351 (2017).</li><li>Prasanphanich, A. F., White, D. E., Gran, M. A., Kemp, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Modeling+of+ABCG2+Transporter+Heterogeneity:+A+Quantitative,+Single-Cell+Analysis+of+the+Side+Population+Assay.">Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay.</a> PLoS Computational Biology. 12 (11), 1005188 (2016).</li><li>Han, 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=An+endogenous+inhibitor+of+angiogenesis+inversely+correlates+with+side+population+phenotype+and+function+in+human+lung+cancer+cells.">An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.</a> Oncogene. 33 (9), 1198-1206 (2014).</li><li>Loebinger, M. R., Sage, E. K., Davies, D., Janes, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAIL-expressing+mesenchymal+stem+cells+kill+the+putative+cancer+stem+cell+population.">TRAIL-expressing mesenchymal stem cells kill the putative cancer stem cell population.</a> British Journal of Cancer. 103 (11), 1692-1697 (2010).</li><li>Chiba, T., 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+purified+from+hepatocellular+carcinoma+cells+harbors+cancer+stem+cell-like+properties.">Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.</a> Hepatology. 44 (1), 240-251 (2006).</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+"side+population"+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells.">A distinct "side population" of cells with high drug efflux capacity in human tumor cells.</a> Proceedings of the National Academy of Science of the United States of America. 101 (39), 14228-14233 (2004).</li><li>Chen, A. Y., Yu, C., Gatto, B., Liu, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+minor+groove-binding+ligands:+a+different+class+of+mammalian+DNA+topoisomerase+I+inhibitors.">DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors.</a> Proceedings of the National Academy of Science of the United States of America. 90 (17), 8131-8135 (1993).</li></ol>

There are several key points to keep in mind for the SP assay. The first is the selection of a proper blocker, such as Verapamil or Reserpine, for each cell line, because the &#34;gate&#34; location of the SP cells is determined according to the position at which a large number of SP cells disappear after the addition of the blocker. For the MDA-MB-231 cell line, Reserpine works well. However, for other cell lines, different blockers might work better.
The second is the concentration of Hoechs...

Cancer stem cells (CSCs) are subsets of cells with self-renewal ability and multiple differentiation potential, which play a vital role in tumor growth, metastasis, and recurrence1,2. Currently, CSCs have been identified to exist in a variety of malignant tumors, including lung, brain, pancreas, prostate, breast, and liver cancers3,4,5,6,7,8,9. Identification of CSCs in these tumors is mainly based on the presence of surface marker proteins, such as high and/or low expression of CD44, CD24, CD133, and Sca-19,10, but a unique marker that can distinguish CSCs from non-CSCs has not been reported so far. Currently, several techniques are used to identify and purify CSCs in tumor tissue or tumor cell lines. These techniques are designed based on the specific properties of CSCs. Among them, assays and sorting of side population (SP) cells are two of the commonly used methods.
SP cells were originally discovered by Goodell et al.11, when they characterized hematopoietic stem cells in mouse bone marrow cells. When the mouse bone marrow cells were labeled with the fluorescent dye Hoechst 33342, a small group of Hoechst 33342 dimly-stained cells appeared in the two-dimensional dot plot of a flow cytometry assay. Hoechst 33342 is a DNA-binding dye and has at least two binding modes that lead to different spectral characteristics. When viewing fluorescence emission at two wavelengths at the same time, multiple populations can be revealed12. In their assay, the Hoechst 33342 was excited at 350 nm and the fluorescence was measured by using the 450/20 nm band-pass (BP) filter and 675 nm edge filter long-pass (EFLP)11. Compared with whole population of bone marrow cells, this group of cells was enriched with hematopoietic stem cells called SP cells11. SP cells are capable of rapidly expelling Hoechst 33342. The efflux of this dye is related to ATP-binding cassette (ABC) transporters13, which can be inhibited by some agents such as Fumitremorgin C14, Verapamil and Reserpine15,16. After that, different proportions of SP cells were detected in a variety of tissues, organs, tumor tissues, and tumor cell lines17,18,19. These SP cells have many characteristics of stem cells17,19.
This manuscript describes Hoechst 33342 labeling and staining of cultured tumor cells and the analysis of SP cells by flow cytometry. Moreover, optimization of the Hoechst 33342 concentration and the proper blocker selection for a specific tumor cell line using this approach are shown. Finally, the effects of stemness promotion or inhibition signals on the proportion of SP in tumor cells are demonstrated. The experimental examples demonstrate that analysis of SP can be used to explore the effects of various signals, such as gene expression, small inhibitors, activators, cytokines, and chemokines, on tumor stemness. Compared to other methods for isolation and purification of CSCs, such as sorting of CD44+/CD24&#8211; population, aldehyde dehydrogenase (ALDH) analysis, and tumor sphere formation assays, this method is easier for manipulation and is cost-effective.

<table>
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
			<td>9.5 cm2 (approx.)</td>
		</tr>
		<tr>
			<td>Colivelin</td>
			<td>MCE</td>
			<td>HY-P1061A</td>
			<td>Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Arg-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries (BIOIND)</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSRFortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer software</td>
			<td>BD Biosciences</td>
			<td>FACSDiva</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry analysis software</td>
			<td>BD Biosciences</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>bisBenzimide H 33342 trihydrochloride</td>
		</tr>
		<tr>
			<td>Polystyrene round bottom test tube</td>
			<td>CORNING</td>
			<td>352054</td>
			<td>12 x 75 mm, 5mL</td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>Sigma-Aldrich</td>
			<td>83580</td>
			<td>(3&#946;, 16&#946;, 17&#945;, 18&#946;, 20&#945;)-11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester</td>
		</tr>
		<tr>
			<td>SKLB816</td>
			<td>Provided by Dr. Shengyong Yang, Sichuan University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td>5-[N-(3,4-Dimethoxyphenylethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride</td>
		</tr>
	</tbody>
</table>

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.

Four experimental SP analyses were performed according to this method. In the first one, we detected the proportion of SP cells in MDA-MB-231, which is a triple negative human breast cancer cell line, under normal conditions. After cell counting, Hoechst 33342 was added into one tube containing 1 x 106 cells to a final concentration of 3 &#181;g/mL. Reserpine and Hoechst 33342 were added to another tube to&#160;final concentrations of 40 &#181;M and 3 &#181;g/mL, respectively. PI was added to both tubes. The d...

Watch this Scientific Journal Video about Analysis of Side Population in Solid Tumor Cell Lines at JoVE.com

Analysis of Side Population in Solid Tumor Cell Lines

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 ...

Analysis of side population cells is one of the commonly used methods to isolate and identify cancer stem cells from tumor tissues and tumor cell lines. This assay is convenient, fast, and cost-effective. This method can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on stemness properties of tumor cells.Many factors can influence the percentage of SP cells in this analysis, so it is critical to explore the proper conditions before the formal experiment. Seed tumor cells in a six-well plate, and incubate them in a 37-degree Celsius incubator supplied with 5%carbon dioxide. When the cell density reaches about 90%aspirate the culture medium, and wash the cells twice with three milliliters of PBS.Then, add 500 microliters of 0.25%trypsin-EDTA to each well, and incubate the plate at 37 degrees Celsius for one to three minutes. Gently tap the plate to detach the cells, add three milliliters of PBS supplemented with 2%FBS to each well, and pipette the cells up and down three to five times to disperse the cell clumps. Examine the cell suspension under the microscope.If cell clumps are present, pass the suspension through a 70-micrometer strainer. Transfer the cells to a 15-milliliter centrifuge tube, and centrifuge them at 200 times g for five minutes. Remove the supernatant, and resuspend the cells in three milliliters of PBS supplemented with 2%FBS, pipetting the cells up and down three to five times to mix.Count cells under the microscope using a hemocytometer, and dilute them in PBS supplemented with 2%FPS to a final concentration of one times 10 to the six cells per milliliter. Prepare two five-milliliter, polystyrene, round-bottom test tubes, and add one milliliter of the cell suspension to each tube. Label one tube as a test tube and the other as a blocker control tube.Prepare a solution of blocker by diluting it to the appropriate concentration. Add it to the blocker control tube and mix well, then incubate the tube at 37 degrees Celsius for 30 minutes. Shake the tube every 10 minutes.Prepare a solution of Hoechst 33342 by diluting it to the appropriate concentration. Add it to the test tube and blocker control tube separately and mix well, then incubate the tubes at 37 degrees Celsius for 60 minutes. Shake the tubes every 10 minutes.After the incubation, centrifuge the cells for five minutes at 200 times g and four degrees Celsius, then carefully aspirate the supernatant. Resuspend the cells in each tube with one milliliter of ice-cold PBS supplemented with 2%FBS, then pipette the cells up and down to mix. Add one microliter of one-milligram-per-milliliter propidium iodide, or PI, to each tube and mix.Click the Parameters tab in the flow cytometer software. First, choose the parameters of FSC, SSC, Hoechst Blue, Hoechst Red, and PI, respectively. Second, choose the logarithmic scale for the PI parameter.Finally, choose area and width scales for the FSC, SSC, Hoechst Blue, Hoechst Red, and PI parameters, respectively. Run the cells from the test tube first, then run the cells from the blocker control tube using the same voltages and gates. Collect 20 to 100, 000 events from each sample to analyze the percentage of SP cells.This method was used to detect the proportion of SP cells in the MDA-MB-231 human breast cancer cell line. The dot plot of FSC-A versus PI-A showed a population of dead cells, cell debris, and the main population of PI negative cells, which was subjected to further analysis. A single cell population gated from the dot plot of FSC-A versus FSC-W and the dot plot of SSC-A versus SSC-W was used to analyze the proportion of SP cells, which was about 0.9%in untreated cells and about 0.09%in cells treated with reserpine.Different staining concentrations of Hoechst 33342 were tested on MDA-MB-435 cells, and it was determined that two micrograms per milliliter was optimal for SP analysis. Low concentrations made it difficult to distinguish SP cells from other populations, while high concentrations caused SP cells to disappear. Verapamil and reserpine were tested to determine the proper blocker for the MDA-MB-435 cells.About 0.4%of cells expelled the dye after verapamil treatment, but the ratio dropped to about 0.1%after reserpine treatment, demonstrating that reserpine is a more appropriate blocker for this cell line. This protocol was also used to determine the proportion of SP cells in A549 human lung adenocarcinoma cells treated with STAT3 activator colivelin and in T47D human breast cancer cells treated with FRA1 inhibitor SKLB816. This assay provides clues to the regulatory effect of signaling pathways on the features of cancer stem cells and facilitates the discovery of new mechanisms, which can ultimately guide the targeted therapy of tumors.

analysis-of-side-population-in-solid-tumor-cell-lines

Research

JoVE Journal

Cancer Research

4.2K visualizações.  Nankai University. A análise de células populacionais laterais é um dos métodos comumente utilizados para isolar e identificar células-tronco cancerígenas de tecidos tumorais e linhas de células tumorais. Este ensaio é conveniente, rápido e econômico. Este método pode contribuir para uma melhor compreensão do efeito de genes ou outros sinais extracelulares e intracelulares sobre as propriedades de tronco das células tumorais.Muitos fatores podem influenciar o percentual de células de SP nes...