&#160;This work was supported by the grants to J.H. from National Natural Science Foundation of China (No. 81800207). The assistance of Beckman Coulter, Inc. in providing support for flow cytometry and parameter calibration is greatly appreciated.&#160;We thank Jiao Chen of the State Key Laboratory of Oral Diseases, West China Hospital of&#160; Stomatology, Sichuan University, and Yu Qi of Regenerative Medicine Research Center, West China Hospital, Sichuan University, for their assistance in&#160;flow cytometry data acquisition.

The experiments in this study utilized a total of 10 C57BL/6 mice aged between 8 and 12 weeks. Experimental operations were conducted in accordance with a protocol that was approved by the Institutional Animal Care and Use Committee of Sichuan University (#201609309). Experimental materials and the parameters of flow cytometry used in this article are listed in the Table of Materials.
1. Isolation and collection of mouse bone marrow cells
<ol>
	<li>Euthanize mice in strict accordance with the institutional guidelines. To induce unconsciousness in the mouse, administer inhalation anesthetics starting with 2% isoflurane, followed by a gradual increase in dosage to 5% isoflurane until cessation of respiration.</li>
	<li>Dissect the mice in the surgical suite or the Fume cupboard. Clean and sterilize the mice with 70% ethanol.</li>
	<li>Cut the skin and muscle of the leg with sharp scissors completely and dissect the femurs.</li>
	<li>Crush the femur gently with a grinding rod in a mortar and flush out bone marrow cells with DMEM medium until the bone turns white.</li>
	<li>Filter the obtained bone marrow cells with a 100 &#181;m filter into 15 mL tubes. Centrifuge at 800 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and lyse residual red blood cells with 1 mL of red blood cell lysis buffer for 1 min at 4 &#176;C. Centrifuge at 800 x g for 5 min at 4 &#176;C. After centrifugation, wash again with DMEM medium.</li>
	<li>Resuspend the cells in 2 mL of incubation solution (DMEM medium + 5% FBS), count the cells with an automatic cell counter and adjust the cell concentration to 1.0 x 106 cells/mL with incubation solution.</li>
</ol>
2. Cellular staining
<ol>
	<li>Supplement 2 mL of bone marrow cell suspension from each mouse with 5 &#181;g/mL Hoechst 33342. To another 1 mL aliquot, add 100 &#181;M verapamil as a negative control.</li>
	<li>Incubate in a water bath at 37 &#176;C for 90 min with gentle agitation every 30 min.</li>
	<li>After incubation, cool the cells on ice for 5 min and then centrifuge at 250 x g for 5 min at 4 &#176;C. Resuspend the cells in a cold running solution (HBSS + 2% FBS). Centrifuge at 4 &#176;C for 5 min and discard the supernatant.</li>
	<li>Resuspend the resulting cell pellet in 500 &#181;L of running solution per sample. Prior to FCM analysis, supplement cells with 2 &#181;g/mL of propidium iodide (PI) and place on ice for approximately 5 min.</li>
</ol>
3. Flow cytometry
NOTE: For a summary of the various systems and configurations used, please see Table 1.
<ol>
	<li>Calibrate the Coefficient of variation (CV) values of the required channels using daily quality control (QC) fluorospheres in the FCMs and perform sample testing following the successful completion of quality control.
	<ol>
		<li>Select the desktop shortcut of software and launch the software.</li>
		<li>Select Start QC/Standardization in the QC/Standardization menu to access the QC experiment.</li>
		<li>Insert the QC fluorospheres sample tube into the tube holder.</li>
		<li>Select Start to load the sample and begin to run the QC procedure. FCMs are ready for use after QC passes.</li>
	</ol>
	</li>
	<li>Excite the Hoechst 33342 dye of the same samples with a 355 nm, 375 nm, and 405 nm laser, respectively, for detection of Hoechst red in the 690/50 nm channel and Hoechst blue in the 450/50 nm channel.
	<ol>
		<li>Create a new experiment by selecting New Experiment in the File menu, specify the file path, and save the experiment.</li>
		<li>Select Set Channel in the Settings menu. Select the channel signal check box (Y585, V450, V660, NUV450, NUV660, UV450, UV660), then add the reagent name in the Label column (Y585: PI; V450: Hoechst Blue-405 nm; V660: Hoechst red-405 nm; NUV450: Hoechst Blue-375 nm; NUV660: Hoechst Blue-375 nm; UV450: Hoechst Blue-355nm; UV660: Hoechst Blue-355 nm).</li>
		<li>Click Pseudo Color Plots icons in the plot area to create plots. Select an axis name to change which channel is displayed.</li>
		<li>Click Add Tube in the Test Tube screen to create new sample tubes and change their names.</li>
		<li>Select Run to load the sample, view the plots, and establish the gates. Adjust the gain and threshold settings. Select Record to save the data.</li>
	</ol>
	</li>
	<li>Design the gate setting logic.
	<ol>
		<li>For the first plot, click the X-axis to select FSC-W and click the Y-axis to select FSC-A. Select Polygon Gate to draw gate A to circle the individual cell and exclude adherent cells (Figure 1A).</li>
		<li>For the second plot, click the X-axis to select FSC-A and click the Y-axis to select SSC-A. Select Polygon Gate to draw gate B to separate non-fragmentary cells and exclude cellular debris (Figure 1B).</li>
		<li>For the third plot, click the X-axis to select PI-A and click the Y-axis to select SSC-A. Select the Polygon Gate to draw gate D. Select Live cells exhibiting negative PI (Figure 1C) and draw gate C to obtain live cells.</li>
		<li>For the fourth two-dimensional plot, click the X-axis to select Hoechst Red and click the Y-axis to select Hoechst Blue. Right-click the Plot and select Property from the drop-down menu. Select Linear Format for both the X-axis and Y-axis. Select Polygon Gate to draw gate SP to get SP cells (Figure 1D).</li>
	</ol>
	</li>
	<li>To assess sample eligibility, use 355 nm of a specific flow cytometer23 for SP cell detection.</li>
	<li>Use the 355 nm (laser power: 20 mW) and 405 nm (laser power: 80 mW) lasers simultaneously to detect both the control cells (with added verapamil) and experimental cells.</li>
	<li>Acquire fluorescence signals at the 690/50 nm and 450/50 nm channels corresponding to the two lasers. Observe the effective stimulation of Hoechst 33342 dye by 355 nm and 405 nm lasers with clear SP cells (Figure 2B-C).</li>
	<li>For the same samples, use the 375 nm (laser power: 60 mW) and 405 nm (laser power: 80 mW) lasers for cell detection.</li>
</ol>

Alternative Laser Excitation Method for Easy Side Population Cell Detection

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	<li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183 (4), 1797-1806 (1996).</li><li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nat Med. 7 (9), 1028-1034 (2001).</li><li>Camargo, F. D., Chambers, S. M., Drew, E., McNagny, K. M., 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=Hematopoietic+stem+cells+do+not+engraft+with+absolute+efficiencies.">Hematopoietic stem cells do not engraft with absolute efficiencies.</a> Blood. 107 (2), 501-507 (2006).</li><li>Challen, G. A., Boles, N. C., Chambers, S. M., 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=Distinct+hematopoietic+stem+cell+subtypes+are+differentially+regulated+by+TGF-beta1.">Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.</a> Cell Stem Cell. 6 (3), 265-278 (2010).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, 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=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J Cell Biol. 159 (1), 123-134 (2002).</li><li>Terrace, J. 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=Side+population+cells+in+developing+human+liver+are+primarily+haematopoietic+progenitor+cells.">Side population cells in developing human liver are primarily haematopoietic progenitor cells.</a> Exp Cell Res. 315 (13), 2141-2153 (2009).</li><li>Martin, J., 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=Adult+lung+side+population+cells+have+mesenchymal+stem+cell+potential.">Adult lung side population cells have mesenchymal stem cell potential.</a> Cytotherapy. 10 (2), 140-151 (2008).</li><li>Challen, G. A., Bertoncello, I., Deane, J. A., Ricardo, S. D., Little, M. 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M., Ng, A. V., Lam, S., Hung, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells.">Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.</a> Cancer Res. 67 (10), 4827-4833 (2007).</li><li>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>Haraguchi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+side+population+of+cancer+cells+from+human+gastrointestinal+system.">Characterization of a side population of cancer cells from human gastrointestinal system.</a> Stem Cells. 24 (3), 506-513 (2006).</li><li>Zhou, J., 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=Activation+of+the+PTEN/mTOR/STAT3+pathway+in+breast+cancer+stem-like+cells+is+required+for+viability+and+maintenance.">Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance.</a> Proc Natl Acad Sci U S A. 104 (41), 16158-16163 (2007).</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 Sci. 107 (4), 433-443 (2016).</li><li>Wang, 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=ALCAM+regulates+multiple+myeloma+chemoresistant+side+population.">ALCAM regulates multiple myeloma chemoresistant side population.</a> Cell Death Dis. 13 (2), 136 (2022).</li><li>Wang, 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=Homoharringtonine+synergizes+with+quizartinib+in+FLT3-ITD+acute+myeloid+leukemia+by+targeting+FLT3-AKT-c-Myc+pathway.">Homoharringtonine synergizes with quizartinib in FLT3-ITD acute myeloid leukemia by targeting FLT3-AKT-c-Myc pathway.</a> Biochem Pharmacol. 188, 114538 (2021).</li><li>Dong, X. 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No conflicts of interest declared.

We used the protocol described to conduct three experiments, with each trial involving 3-4 mice, resulting in a total of 10 mice. The proportion of SP cells ranged from 0.05% to 0.76%. It is important to note that individual variations were observed among the mice. We utilized four flow cytometers to analyze the Hoechst samples. It is observed that the excitation of Hoechst dye by a 20 mW 355 nm laser on the new version of flow cytometry is equivalent to that of a 100 mW 355 nm laser on old-fashioned flow cytometry. This...

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The SP cells are characterized by the pumping of the fluorescent DNA dye out of the cells through their ATP-binding cassette (ABC) transporter1,2. The method was originally established for isolating murine bone marrow hematopoietic stem cells (HSCs)1. The bone marrow SP cells were enriched with a population of HSCs characterized by &#160;CD117+Sca-1+Lin-Thy1low&#160;expression3,4. Thereafter, the method was widely used to isolate and enrich stem cells from other tissues, including cardiac muscle5, liver6, lung7, kidney8, and forebrain9. In particular, the method has been applied to isolate cancer stem cells (CSCs) in the past decade. CSCs represent a small population of cells that possess the properties of tumor initiation, self-renewal, resistance to chemotherapy, and metastatic potential10. CSCs were initially identified in hematopoietic malignancies11 and subsequently observed in various solid tumors such as the prostate12, ovarian13, gastric14, breast15, and lung16 carcinomas. Despite the availability of various techniques for CSC identification, the SP technique remains a favored choice owing to its broad applicability across diverse tissues and cell lines. Moreover, it is a valuable technique that isolates CSCs using fluorescence-activated cell sorting (FACS)16,17,18. The experimental findings demonstrate that SP cells exhibit pronounced tumorigenicity and display elevated expression levels of stem cell-associated genes19,20. Our previous study21 has also demonstrated that transcriptomic analysis of isolated SP cells in multiple myeloma reveals enrichment of signaling pathways associated with stem cells, such as the hedgehog pathway. Meanwhile, we performed pathway enrichment analyses on the genes differentially expressed in the SP cells of acute myelogenous leukemia22. The altered genes were enriched in stem cell-related pathways (Wnt/&#946;-catenin, TGF-&#946;, Hedgehog, Notch). We found that 1 x 105 SP cells could form tumors in BALB/c null mice22, whereas non-SP cells could not, indicating that SP cells had characteristics of leukemia stem cells. The efficacy of the Hoechst SP method in the identification of CSCs is evident.
The Hoechst SP protocol has been refined and enhanced through the progression of research. The protocol entails stringent control of the dye concentration, cell density, incubation temperature and duration, buffer composition, and pH value. The cell samples prepared in accordance with this protocol were subjected to flow cytometric analysis. Due to the excitation of Hoechst dye with a UV laser at 355 nm and detection of its fluorescence emission using both a 690/50 nm filter (Hoechst Red) and 450/50 nm filter (Hoechst Blue), a 350 nm laser is required for FCM. However, 350 nm lasers are not commonly equipped in most FCMs because of their high cost. Hence, we attempted to find an alternate approach for&#160;dye excitation for the effective detection of SP cells by flow cytometry. In this study, the capability of the 375 nm and 405 nm lasers in detecting SP cells was assessed and compared with that of the 355 nm laser. Our findings demonstrate a remarkable similarity between the SP cells detected by the 355 nm, 375 nm, and 405 nm lasers. These results suggest that the high-power 375 nm and 405 nm lasers can serve as feasible alternatives to the 355 nm laser for SP cell detection. The inclusion of additional excitation light sources for Hoechst 33342 facilitates the use of more flow cytometry models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic cell counter</td>
			<td>Countstar</td>
			<td>1M1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Filter(100 &#181;m)</td>
			<td>BIOFIL</td>
			<td>CSS-013-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Daily quality control fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>B5230</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5g/L glucose (DMEM medium)</td>
			<td>CORNING</td>
			<td>10-013-CVRC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>CORNING</td>
			<td>35-081-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Hyclone</td>
			<td>SH30030.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In Figure 2, the control group was treated with verapamil, which blocks ABC transporters in stem cells to prevent the elimination of Hoechst 33342. Thereby, the stem cells in the non-verapamil group expel Hoechst 33342 and form a negative cell population known as SP cells. The 355 nm laser effectively excited the Hoechst 33342 dye, resulting in clear observation of SP cells of bone marrow (Figure 2A). The SP cells of the same samples detected by the 355 nm laser...

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.

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 ...

effective-detection-of-hoechst-side-population-cells-by-flow-cytometry

Research

JoVE Journal

Cancer Research

582 Views.  Sichuan University. 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.

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

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.

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 ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

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 ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...