Name: Analysis of Side Population in Solid Tumor Cell Lines (Video) | JoVE Journal
Uploaded: 2021-02-23T13:00:00.000+00:00
Duration: 6 min 26 s
Description: Learn a convenient method to measure side population cells in solid tumor cell lines for cancer stem cell research. This cost-effective assay helps isolate cancer stem cells and study tumor biology.

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5 minutes

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 stemness features of tumor cells, tumor cells are seeded in a 6 well plate and pretreated with inhibitors or activators for a certain amount of time before harvest.</li>
		<li>Add 500 &#181;L of 0.25% trypsin-EDTA to each well of the 6 well plate, place the plate in an incubator at 37 &#176;C for 1&#8211;3 minutes (min), and gently tap the plate to detach the cells. 
		NOTE: Prolonged digestion will affect the SP profile due to changes in cell viability.</li>
		<li>Add 3 mL of PBS supplemented with 2% fetal bovine serum (FBS) to each well of the 6 well plate&#160;to terminate the digestion and gently pipette the cells up and down 3&#8211;5x to disperse the cell clumps.</li>
		<li>Add 0.5 mL of cell suspension to one well of a new 6 well plate and examine it under a microscope. If cell clumps are observed, pass the cell suspension through a 70 &#181;M cell strainer. 
		NOTE: This is an optional step.</li>
		<li>Transfer the cells to a 15 mL&#160;centrifuge tube. Centrifuge cells at 200 x g for 5 min to pellet cells. Remove the supernatant and resuspend them in 3 mL of PBS supplemented with 2% FBS. Pipette the cells up and down 3&#8211;5x to mix thoroughly.</li>
	</ol>
	</li>
	<li>Cell counts
	<ol>
		<li>Add 50 &#956;L of the cell suspension into a 1.5 mL microcentrifuge tube and mix it with 50 &#956;L trypan blue solution. Pipette 10 &#956;L of the mixture to count the number of living cells using a standard method, such as a hemocytometer.</li>
		<li>Dilute the cells in PBS supplemented with 2% FBS to a final concentration of 1 x 106 cells/mL. Add 1 mL of the cell suspension to a 5 mL polystyrene round bottom test tube and prepare two sample tubes.</li>
	</ol>
	</li>
</ol>
2. Cell staining with Hoechst 33342
<ol>
	<li>Add Hoechst 33342 to one tube to reach an appropriate final concentration. 
	NOTE: For example, the appropriate concentration of Hoechst 33342 is 3 &#181;g/mL for MDA-MB-231 cells.</li>
	<li>Add a blocker&#160;(e.g., Fumitremorgin C, Verapamil, or Reserpine) to another tube to an appropriate final concentration and incubate the tube at 37 &#176;C for 30 min before adding the same concentration of Hoechst 33342 as described in step 2.1. 
	NOTE: For MDA-MB-231 cells, the appropriate blocker is Reserpine (40 &#181;M). Reserpine is used as a blocking control to verify the absence of cells in the gated SP area.</li>
	<li>Prepare several tubes containing cells and add Hoechst 33342 to&#160;different concentration gradients. After a flow cytometry assay, choose the proper concentration according to the profile and proportion of SP. 
	NOTE: This is an optional step used to define the proper concentration of Hoechst 33342.</li>
	<li>Prepare several tubes containing cells, add the blocker to different concentration gradients, incubate the tubes at 37 &#176;C for 30 min, then add Hoechst 33342 to an appropriate concentration. After the flow cytometry assay, choose the proper concentration according to the absence of cells in the gated SP area. 
	NOTE: This is an optional step used to define the proper concentration of blocker.</li>
	<li>Place the tubes in a 37 &#176;C incubator and incubate them for 60 min, shaking the tubes every 10 min. 
	NOTE: Shaking tubes thoroughly is important for staining, because it ensures complete contact between the cells and the dye for better staining results.</li>
	<li>After the incubation, centrifuge the cells at 200 x g, 4 &#176;C for 5 min, and aspirate the supernatant carefully, because the cells form&#160;very loose and unstable pellets.</li>
	<li>Resuspend cells of each tube in 1 mL of ice-cold PBS supplemented with 2% FBS and pipette the cells up and down 3&#8211;5x to mix thoroughly. Add 1 &#956;L of 1 mg/mL propidium iodide (PI) to the suspension of each tube to identify dead cells. 
	NOTE: All procedures in this step should be performed at 4 &#176;C to inhibit the efflux of Hoechst 33342 from the tumor cells. The tubes should be protected from direct exposure to light.</li>
</ol>
3. Analysis by flow cytometry
NOTE: Instructions for use of the flow cytometer software (see Table of Materials) are described in this section and Supplementary Figures 1&#8211;10.
<ol>
	<li>In the flow cytometer software, click the FOLDER button. Click the Experiment button, then click New Experiment (Supplementary Figure 1A,B).</li>
	<li>Click the OK button. &#8220;Experiment_001&#8221; will show up under the FOLDER (Supplementary Figure 2A,B).</li>
	<li>Click the Experiment_001 button to change the folder name to a specific name (e.g., &#34;20191118-SP&#34;). Click Enter. The new name (&#34;20191118-SP&#34;) will show up under FOLDER (Supplementary Figure 3A,B).</li>
	<li>Click the New Specimen button to add a specimen to the new experiment folder (&#34;20191118-SP&#34;). Click the New Tube button to add a tube to the specimen. Click the Arrowhead button (Supplementary Figure 4A-C).</li>
	<li>Click the Parameters button and set up the parameters of the flow cytometer (Supplementary Figure 5).
	<ol>
		<li>Use a 610 nm dichroic mirror short-pass (DMSP) to separate the emission wavelengths. Use a 450/20 nm BP filter to collect the blue fluorescence and a 675 nm EFLP to collect the red fluorescence. 
		NOTE: Hoechst 33342 is excited with a UV laser at 355 nm and PI is excited at 488 nm.</li>
	</ol>
	</li>
	<li>Run the cell samples on the flow cytometer. 
	NOTE: SP cells can be sorted by fluorescence-activated cell sorting (FACS) under sterile conditions. A total of 100,000&#8211;500,000 cells should be collected for the follow-up experiments.
	<ol>
		<li>Run cells stained with Hoechst 33342 on the flow cytometer.
		<ol>
			<li>Place tubes containing cells stained with Hoechst 33342 on the cytometer.</li>
			<li>Click the Dot Plot button to display the dot plot, then click the X-axis and set it to &#8220;FSC-A&#8221;; click the Y-axis and set it to &#8220;PI-A&#8221;. Display the dot plot of forward scatter pulse area (FSC-A, X-axis set to linear mode) versus the PI fluorescence (Y-axis set to logarithmic scale). Adjust the voltages to show the living cells in the right side and the non-living cells, which are brightly&#160;stained with PI, in the upper left&#160;corner. Then, establish a polygon gate to exclude dead cells and cell debris (Figure 1A) by clicking the Polygon Gate button to gate the P1 subset, also known as FSC-A, PI-A subset (Supplementary Figure 6A,B).</li>
			<li>Click the Dot Plot button to display the dot plot, click the X-axis and set it to &#8220;FSC-A&#8221;; click the Y-axis and set it to &#8220;FSC-W&#8221;. Display the dot plot of FSC-A (X-axis) versus forward scatter pulse width (FSC-W, Y-axis). Right-click the dot plot, click the P1 button under the Show Populations button. Click the Rectangular Gate button to create a rectangular gate to gate the P2 subset (also known as FSC-A, FSC-W subset). This will exclude cells with large volumes (Supplementary Figure 7A-C and Figure 1A).</li>
			<li>Click the Dot Plot button to display the dot plot, click the X-axis and set it to &#8220;SSC-A&#8221;; click the Y-axis and set it to &#8220;SSC-W&#8221;. Display the dot plot of side scatter pulse area (SSC-A, X-axis) versus side scatter pulse width (SSC-W, Y-axis). Right-click the dot plot, and click the P2 button under Show Populations button. Then click the Rectangular Gate button to create a rectangular gate to gate the P3 subset (also known as SSC-A, SSC-W subset). This will obtain a cell population with uniform granularity (Supplementary Figure 8A-C and Figure 1A).</li>
			<li>Click the Dot Plot button to display the dot plot, click the X-axis and set it to &#8220;Hoechst Red-A&#8221;; click the Y-axis and set it to &#8220;Hoechst Blue-A&#8221;. Display the dot plot of Hoechst Red-A (X-axis) versus Hoechst Blue-A (Y-axis). Right-click the dot plot, click the P3 button under the Show Populations button. Click the Polygon Gate button to create a polygon gate to gate the P4 subset (also known as Hoechst Red-A, Hoechst Blue-A subset). Right-click the dot plot, click the Show Population Hierarchy button to show the population hierarchy. 
			NOTE: The dot plot will show three different populations: 1) a G0-G1 phase population near the center of the graph; 2) a S-G2/M phase population near the upper right corner; 3) the SP. The SP is then gated for further analysis (Supplementary Figure 9A-D and Figure 1A). If the dot plot of Hoechst Red-A versus Hoechst Blue-A does not show a&#160;SP profile similar to that shown in Figure 1A, the voltages should be adjusted until a similar profile is seen. Meanwhile, adjust all of above gates accordingly.</li>
			<li>After determining the gate region of the SP cells, click Acquire Data to collect 20,000&#8211;100,000 events from each sample to analyze the percentage of SP cells (Supplementary Figure 10).</li>
		</ol>
		</li>
		<li>Run the Hoechst 33342-stained cells treated with blocker on the flow cytometer.
		<ol>
			<li>Place tubes containing blocker on the cytometer and run cells using the same voltages and gates to further check whether the voltages and gates are selected appropriately. 
			NOTE: Compared with Hoechst 33342 staining alone, only&#160;a very small proportion of cells, if any, should appear in the gated area of the SP (Figure 1B).</li>
			<li>Collect 20,000&#8211;100,000 events from each sample to analyze the percentage of SP cells.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Data analysis
NOTE: Instructions for the use of the flow cytometry analysis software (see Table of Materials) are described in this section and Supplementary Figures 11&#8211;16.
<ol>
	<li>Copy the files in fcs format to a computer, open the flow cytometry analysis software, and drag one sample file to the software (Supplementary Figure 11A,B).</li>
	<li>Gate cells and obtain the percentage of SP cells.
	<ol>
		<li>Double click this sample file, click the X-axis and set it to &#8220;FSC-A&#8221;; click the Y-axis and set it to &#8220;PI-A&#8221;. Then, create a polygon gate and click the OK button to obtain the FSC-A, PI-A subset (Supplementary Figure 12A-E).</li>
		<li>Double click the FSC-A, PI-A subset file, click the X-axis and set it to &#8220;FSC-A&#8221;; click the Y-axis and set it to &#8220;FSC-W&#8221;. Then, create a rectangular gate and click the OK button to obtain the FSC-A, FSC-W subset (Supplementary Figure 13A-E).</li>
		<li>Double click the FSC-A, FSC-W subset file, click the X-axis and set it to &#8220;SSC-A&#8221;; click the Y-axis and set it to &#8220;SSC-W&#8221;. Then, create a rectangular gate and click the OK button to obtain the SSC-A, SSC-W subset (Supplementary Figure 14A-E).</li>
		<li>Double click the SSC-A, SSC-W subset file, click the X-axis and set it to &#8220;Hoechst Red-A&#8221;; click the Y-axis and set it to &#8220;Hoechst Blue-A&#8221;. Then, create a polygon gate and click the OK button to obtain the Hoechst Red-A, Hoechst Blue-A subset (Supplementary Figure 15A-E).</li>
		<li>Open the Layout Editor by clicking the Open Layout Editor button. Drag the SSC-A, SSC-W subset file to Layout Editor, then click the Click to save layout window to file button to save the image results (Supplementary Figure 16A-C).</li>
	</ol>
	</li>
	<li>Save the workspace to keep the gating information when closing the software.</li>
	<li>Perform t-test analyses with statistical analysis software to compare the difference between two groups. A value of P &#60; 0.05 was defined as statistically significant.</li>
</ol>

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<li>Shen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ICAM3+mediates+inflammatory+signaling+to+promote+cancer+cell+stemness%5BTitle%5D)+AND+%22Cancer+Letters%22%5BJournal%5D)">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/pubmed?term=((Baicalein+Suppresses+Stem+Cell-Like+Characteristics+in+Radio-+and+Chemoresistant+MDA-MB-231+Human+Breast+Cancer+Cells+through+Up-Regulation+of+IFIT2%5BTitle%5D)+AND+%22Nutrients%22%5BJournal%5D)">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/pubmed?term=((Entrapped+doxorubicin+nanoparticles+for+the+treatment+of+metastatic+anoikis-resistant+cancer+cells%5BTitle%5D)+AND+%22Cancer+Letters%22%5BJournal%5D)">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/pubmed?term=((ADAM23+is+downregulated+in+side+population+and+suppresses+lung+metastasis+of+lung+carcinoma+cells%5BTitle%5D)+AND+%22Cancer+Science%22%5BJournal%5D)">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/pubmed?term=((The+mechanisms+for+lung+cancer+risk+of+PM2.5+%3A+Induction+of+epithelial-mesenchymal+transition+and+cancer+stem+cell+properties+in+human+non-small+cell+lung+cancer+cells%5BTitle%5D)+AND+%22Environmental+Toxicology%22%5BJournal%5D)">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/pubmed?term=((Kinetic+Modeling+of+ABCG2+Transporter+Heterogeneity%3A+A+Quantitative%2C+Single-Cell+Analysis+of+the+Side+Population+Assay%5BTitle%5D)+AND+%22PLoS+Computational+Biology%22%5BJournal%5D)">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/pubmed?term=((An+endogenous+inhibitor+of+angiogenesis+inversely+correlates+with+side+population+phenotype+and+function+in+human+lung+cancer+cells%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">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/pubmed?term=((TRAIL-expressing+mesenchymal+stem+cells+kill+the+putative+cancer+stem+cell+population%5BTitle%5D)+AND+%22British+Journal+of+Cancer%22%5BJournal%5D)">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/pubmed?term=((Side+population+purified+from+hepatocellular+carcinoma+cells+harbors+cancer+stem+cell-like+properties%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">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/pubmed?term=((A+distinct+%22side+population%22+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((DNA+minor+groove-binding+ligands%3A+a+different+class+of+mammalian+DNA+topoisomerase+I+inhibitors%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+of+the+United+States+of+America%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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 cm&lt;sup&gt;2&lt;/sup&gt; (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></td><td>BD LSRFortessa</td></tr><tr><td>Flow cytometer software</td><td>BD Biosciences</td><td></td><td>FACSDiva</td></tr><tr><td>Flow cytometry analysis software</td><td>BD Biosciences</td><td></td><td>FlowJo</td></tr><tr><td>Hoechst 33342</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, 5 mL</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&amp;beta;, 16&amp;beta;, 17&amp;alpha;, 18&amp;beta;, 20&amp;alpha;)-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-[&lt;em&gt;N&lt;/em&gt;-(3,4-Dimethoxyphenylethyl)methylamino]
-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride</td></tr></tbody></table>

analysis of side population in solid tumor cell lines

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 µg/mL. Reserpine and Hoechst 33342 were added to another tube to final concentrations of 40 µM and 3 µg/mL, respectively. PI was added to both tubes. The dot plot of FSC-A (X-...

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-in-solid-tumor-cell-lines

Research

JoVE Journal

Cancer Research

The following protocol describes a method for isolating the side population of T-cell Acute Lymphoblastic Leukemia (T-ALL) tumor cells, consisting of stem cells, from an adult Zebrafish. The side population lacks prominent cell markers and hence we use Hoechst 33342 dye efflux assay to isolate them.

Side Population Assay: A Method to Isolate Side Population of Tumor Cells from Zebrafish

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.

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

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

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).

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

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.

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

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.

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

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

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.

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Learn a convenient method to measure side population cells in solid tumor cell lines for cancer stem cell research. This cost-effective assay helps isolate cancer stem cells and study tumor biology.

Analysis of Side Population in Solid Tumor Cell Lines

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