This work was supported by the University of Chicago Women's Board, the Wendy Will Case Fund, and the University of Chicago Comprehensive Cancer Center Support Grant (P30 CA014599). We would like to thank the Flow Cytometry Core at the University of Chicago for their help in setting up this experiment, and well as other members of the de Jong lab, particularly Leslie Pedraza and Sean McConnell.

All procedures with zebrafish have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago. The University of Chicago is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
1. Generating and Isolating Fluorescently Labeled T-ALL Cells in Zebrafish
<ol>
	<li>Microinjection into syngeneic zebrafish embryos
	<ol>
		<li>Using commercially available kits, isolate circular rag2:c-myc and rag2...

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+human+disease.">Zebrafish: a model system for the study of human disease.</a> Curr Opin Genet Dev. 10 (3), 252-256 (2000).</li><li>White, R., Rose, K., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cancer:+the+state+of+the+art+and+the+path+forward.">Zebrafish cancer: the state of the art and the path forward.</a> Nat Rev Cancer. 13 (9), 624-636 (2013).</li><li>Kobayashi, I., 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+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111 (3), 1131-1137 (2008).</li><li>Tsinkalovsky, O., Vik-Mo, A. O., Ferreira, S., Laerum, O. D., Fjose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+kidney+marrow+contains+ABCG2-dependent+side+population+cells+exhibiting+hematopoietic+stem+cell+properties.">Zebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stem cell properties.</a> Differentiation. 75 (3), 175-183 (2007).</li><li>Langenau, D. 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=Myc-induced+T+cell+leukemia+in+transgenic+zebrafish.">Myc-induced T cell leukemia in transgenic zebrafish.</a> Science. 299, 887-890 (2003).</li><li>Blackburn, J. S., Liu, S., Langenau, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+frequency+of+tumor-propagating+cells+using+limiting+dilution+cell+transplantation+in+syngeneic+zebrafish.">Quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish.</a> J Vis Exp. , e2790 (2011).</li><li>Martelli, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+signaling+pathways+in+T-cell+acute+lymphoblastic+leukemia+initiating+cells.">Targeting signaling pathways in T-cell acute lymphoblastic leukemia initiating cells.</a> Adv Biol Regul. 56, 6-21 (2014).</li><li>Smith, A. 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=High-throughput+cell+transplantation+establishes+that+tumor-initiating+cells+are+abundant+in+zebrafish+T-cell+acute+lymphoblastic+leukemia.">High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.</a> Blood. 115 (16), 3296-3303 (2010).</li><li>Blackburn, J. 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=Clonal+evolution+enhances+leukemia-propagating+cell+frequency+in+T+cell+acute+lymphoblastic+leukemia+through+Akt/mTORC1+pathway+activation.">Clonal evolution enhances leukemia-propagating cell frequency in T cell acute lymphoblastic leukemia through Akt/mTORC1 pathway activation.</a> Cancer Cell. 25, 366-378 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Plasmid+Purification.">Basic Methods in Cellular and Molecular Biology. Plasmid Purification.</a> JoVE Science Education Database. , (2016).</li><li>Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2:+a+versatile+gene+transfer+vector+in+vertebrates.">Tol2: a versatile gene transfer vector in vertebrates.</a> Genome Biol. 8, (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Mizgireuv, I. V., Revskoy, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantable+tumor+lines+generated+in+clonal+zebrafish.">Transplantable tumor lines generated in clonal zebrafish.</a> Cancer Research. 66 (6), 3120-3125 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. , (1993).</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> Proc Natl Acad Sci. USA. 101 (39), 14228-14233 (2004).</li><li>Komuro, 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=Identification+of+side+population+cells+(stem-like+cell+population)+in+pediatric+solid+tumor+cell+lines.">Identification of side population cells (stem-like cell population) in pediatric solid tumor cell lines.</a> J Pediatr Surg. 42 (12), 2040-2045 (2007).</li><li>Moshaver, B., 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+of+a+small+subpopulation+of+candidate+leukemia-initiating+cells+in+the+side+population+of+patients+with+acute+myeloid+leukemia.">Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.</a> Stem Cells. 26 (12), 3059-3067 (2008).</li></ol>

The side population assay is highly sensitive; therefore, small changes to the protocol can result in a difficulty in detecting side population cells. First, the temperature during the staining step is specific to each animal/cell system. For mammalian systems, the side population assay is typically performed at 37 &#176;C2. When the zebrafish T-ALL cells were incubated at 37 &#176;C, many of the cells died, which made this incubation temperature unacceptable (data not shown). When Kobayashi and c...

The side population assay capitalizes on the enhanced ability of certain cells within a tissue to efflux the DNA binding dye Hoechst 33342 due to high levels of the ATP binding cassette (ABC) transporter proteins on the cell membrane. The cells that efflux the Hoechst 33342 dye can be identified using dual wavelength flow cytometric analysis after the dye is excited by a UV laser. This assay was first used to identify murine hematopoietic stem cells (HSCs)1, but it has since been used to identify stem/progenitor cell populations in many tissues and cancers (reviewed in reference 2). However, not all populations of cells have a side population, and not all side populations are enriched for stem/progenitor cells.
The zebrafish is a powerful vertebrate genetic model system for studying human cancer3,4, with a number of advantages over traditional murine models of cancer. Zebrafish embryos are externally fertilized and are optically clear, facilitating transgenesis and the in vivo observation of pathologic processes, including cancer initiation and progression. To date, the side population assay to detect potential stem or progenitor cells has only been applied to the kidney marrow in zebrafish to identify HSCs, and not to any zebrafish cancer models5,6.
The zebrafish model of T-cell acute lymphoblastic leukemia (T-ALL) is morphologically and genetically similar to human T-ALL7,8,11. T-ALL is an aggressive malignancy that, in humans, accounts for 10-15% of pediatric and 25% of adult ALL cases9. While the treatment of T-ALL has improved, relapse is still common and is associated with a poor prognosis. T-ALL tumors are heterogeneous and contain many different tumor cell subpopulations, including leukemia initiating cells (LICs). LICs are defined by their ability to regrow the entire tumor from a single cell, and the frequency of LICs within a tumor cell population can be calculated by transplanting varying cell doses into recipients via a limiting dilution transplantation assay (LDA). While LDA experiments have been performed in zebrafish to calculate the frequency of LICs8,10,11, this determination is made in hindsight and does not allow for the prospective isolation of LICs. Therefore, a method to prospectively isolate a population enriched for cancer stem cell activity is lacking. Identifying and isolating side population cells from zebrafish T-ALLs is the first step towards addressing this deficiency.
The protocol presented here describes how to efficiently generate T-ALL tumors in zebrafish utilizing tol2-mediated transgenesis, harvest the T-ALL tumors cells, and stain them with Hoechst 33342 to identify the side population of cells. Future in vivo experiments with zebrafish T-ALL could include whether the side population cells are enriched for LICs or have other stem- or progenitor-like properties.

<table border="0" cellpadding="0" cellspacing="0" width="859">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Syngeneic zebrafish (CG2)</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td style="width:208px;">See Mizgireuv et al., 2006</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">rag2:c-myc plasmid</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td>Langenau Laboratory</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:245px;">rag2:GFP plasmid constructed in de Jong Lab</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td style="width:208px;">rag2 promoter from Langenau Laboratory</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">pCS2-transposase plasmid</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td>Chien Laboratory</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">NotI -HF restriction enzyme</td>
			<td style="width:199px;">New England Bio-Labs Inc.</td>
			<td>R3189S</td>
			<td>500 units in 25 &#181;L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:245px;">mMessage Machine SP6 Transcription Kit</td>
			<td style="width:199px;">Ambion</td>
			<td style="width:208px;">Thermo Fisher Scientific - AM1340</td>
			<td>25 reactions</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">QIAGEN Plasmid Midi Kit</td>
			<td style="width:199px;">Qiagen, Inc.</td>
			<td style="width:208px;">12643</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">SteREO Discovery V8 Microscope</td>
			<td style="width:199px;">Carl Zeiss Microscopy</td>
			<td>495015-0008-000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Digital microinjector</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-D</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Brass straight-arm needle holder</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Magnetic base and stand</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-HBMB</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Micromanipulator</td>
			<td style="width:199px;">Narishige International</td>
			<td style="width:208px;">MN153</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Glass capillaries</td>
			<td style="width:199px;">Sutter Instrument Co.</td>
			<td style="width:208px;">B100-50-10</td>
			<td>10 cm without filament</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Flaming/Brown micropipette puller</td>
			<td style="width:199px;">Sutter Instrument Co.</td>
			<td style="width:208px;">P-97</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Microloader pipette tips</td>
			<td style="width:199px;">Eppendorf North America Inc.</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Phenol Red solution</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">P0290</td>
			<td>0.5% concentration 100 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Plastic Transfer pipettes</td>
			<td style="width:199px;">Fisherbrand</td>
			<td>137119D</td>
			<td>graduated 7.5 mL bulb</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tricaine methanesulfonate (MS-222)</td>
			<td style="width:199px;">Western Chemical, Inc.</td>
			<td style="width:208px;">Fisher Scientific - NC0342409</td>
			<td>100 g</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Fetal bovine serum (FBS)</td>
			<td style="width:199px;">HyClone Laboratories, Inc.</td>
			<td style="width:208px;">Fisher Scientific - SH3007103</td>
			<td>500 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:199px;">Gibco by Life Technologies</td>
			<td style="width:208px;">1001-023</td>
			<td>pH 7.4 (1x) - 500 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Heparin sodium salt</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:208px;">2106</td>
			<td>15 - 300 unit vials</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">40 um mesh filter</td>
			<td style="width:199px;">Falcon Corning Brand</td>
			<td style="width:208px;">352340</td>
			<td>Case of 50</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Trypan blue</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">T8154</td>
			<td>20 mL - Solution (0.4%)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Hoechst 33342</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:208px;">H3570</td>
			<td>10 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Verapamil</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">V4629</td>
			<td>1 g</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">D8418</td>
			<td>100 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Propidium Iodide</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:208px;">P3566</td>
			<td>10 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">FACSAria Fusion cell sorter</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">BD FACSDiva 8.0.1</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">FlowJo v10.2</td>
			<td style="width:199px;">FlowJo, LLC</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of the side population in myc-induced t-cell acute lymphoblastic leukemia in zebrafish

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

To efficiently generate fluorescent myc-induced T-ALL tumors in zebrafish, circular DNA constructs flanked by tol2 transposase sites can be co-injected with tol2 RNA. Previous studies from the injection of linearized DNA into zebrafish embryos report a 5% transgenesis rate8. With the protocol adjusted to include tol2-mediated transgenesis, transgenesis rates ranging from 10-44% can be observed (Table 1).
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Watch this Scientific Journal Video about Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish at JoVE.com

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

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

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

The overall goal of this procedure is to isolate side population cells from a zebrafish model of myc-induced T-cell acute lymmphoblastic leukemia. The side population assay has been used to identify stem and progenitor cell populations in many normal and cancer tissues. But it hasn't yet been applied to any zebrafish cancer models.The main advantage of this technique is that it allows the prospective isolation of a population of cells in zebrafish T-ALL that may be enriched for cancer stem cell activity. Although the method we describe here has broad applications in zebrafish leukemia research it may also be applicable to other malignant and non-malignant zebrafish cell types. Individuals new to this method may struggle because the method is sensitive to subtle changes.Even though the method seems straightforward, optimization is required for each cell type tested. Prepare 20 milliliters of 10 percent heat-inactivated FBS in 0.9x PBS. Add one milliliter of the FBS/PBS solution to a vial of a heparin sodium salt containing 300 units of the anticoagulant.Then, using an epifluorescent microscope, select fish in which tumor cells have invaded 50 percent of the body, or fish that are identified as having difficulty eating or swimming. Euthanize the fish by transferring it to a solution of one to two milligrams per milliliter tricaine in fish water. Observe the animal for gill movement and heartbeat to confirm euthanasia.After removing the head of the fish, inject with a pipette 100 microliters of heparin solution into its body cavity to remove excess red blood cells. Aspirate all liquid that pours out and pipette it into a 1.5 milliliter microcentrifuge tube. Then, using fresh pipette tips, wash the body cavity two more times or until the washing solution does not contain any blood, and discard the liquid collected from all washes.To collect leukemia cells, inject 100 microliters of FBS/PBS solution into the pre-washed body cavity. Under a dissecting microscope, apply gentle pressure to the body of the fish with a pipette tip to force out the tumor cells. Collect the released liquid into a 1.5 milliliter microcentrifuge tube.Repeat isolation until most of the tumor cells are harvested. Collect all cells in the same 1.5 milliliter microcentrifuge tube and keep the obtained suspension at room temperature. Then, mix the isolated tumor cells by gently pipetting to dissociate cell clumps.Filter the cell suspension through a 40 micrometer mesh filter. And wash the filter one to two times with 100 microliters of FBS/PBS. Once filtered, keep the cells at room temperature.Finally, in a new tube, mix two microliters of the cell suspension with 18 microliters of 0.04 percent sterile Trypan Blue solution. Load 10 microliters of the obtained suspension onto a hemocytometer and count the viable fluorescent tumor cells. It is important to collect a clean sample of T-ALL cells.The concentration of Hoechst three three three four two used here is based on the number of live tumor cells collected. Additional cellular debris or non-tumor cells may interfere with the assay. Prior to cell staining, dilute Hoechst dye in a nuclease-free water to obtain one milligram per milliliter working solution.Store the prepared solution covered with aluminum foil at four degrees Celsius. Then, dissolve 49.1 milligrams of verapamil in one milliliter of DMSO to obtain a 100 micromolar inhibitor solution. Next, prepare the sample by adding FBS/PBS, 2.5 microliters of DMSO, 15 microliters of one milligram per milliliter Hoechst dye, and 10 to the six tumor cells to a fax tube with a final volume of one milliliter.To prepare the control, by adding FBS/PBS, 2.5 microliters of 100 million molar verapamil, 15 microliters of one milligram per milliliter Hoechst dye, add 10 to the six tumor cells with a final volume of one milliliter. Incubate the sample and the control in a 28 degree Celsius water bath in the dark for 120 minutes. After the incubation, place the cells immediately on ice and keep them in the dark.Centrifuge the cells at 300 times G four degrees Celsius for six minutes. Then, remove the supernatint and wash the cells with one milliliter of FBS/PBS. Spin the cells at 300 times G four degrees Celsius for six minutes once more.Once the cells are pelleted remove the supernatint and re-suspend the cells in one milliliter of FBS/PBS. Keep the samples at four degrees Celsius until cell sorting. 15 minutes prior to cell sorting, add two microliters of one milligram per milliliter propidium iodine stock solution per each one milliliter of the cell suspension.Then, vortex the samples well. Analyze the stained zebrafish T-ALL cell suspensions using a fluorescence activated cell sorter. Excite samples with a UV laser for Hoechst dye analysis and a 488 nanometer laser for the detection of propidium iodide or GFP positive T-ALL cells.The number of cells, concentration of dye, incubation temperature, and incubation length may need to be optimized for each different cell type used. And changes to each of these conditions can drastically alter the results of the assay. To identify the side population among T-ALL cells isolated from zebrafish, first cellular debris and cell clumps are excluded.Propidium iodide intake and GFP expression in a single cell population are then analyzed to identify live tumor cells. To visualize the side population, blue and red emissions of the Hoechst dye are then compared. The side population appears as a dim tail of cells extending from the left side of the main cell population towards the blue fluorescence axis.Presented here are results of the side population analysis carried out for two different tumor cell samples. A side population was identified in both specimens. However, different tumor samples contained varied numbers of the side population cells.Regardless of the tumor sample analyzed, treatment of the sample with an efflux inhibitor of verapamil resulted in the disappearance of the signal observed for the previously identified cells further confirming the side population phenotype. Once mastered, the side population technique on a zebrafish T-ALL can be done in less than four hours if it is performed properly and as we presented here. While attempting this procedure, it's important to remember the steps that have been optimized for zebrafish T-ALL such as incubation temperature and time, dye concentration, and efflux inhibitor.These may need to be changed for different experimental systems. Following this procedure, other methods, like RNA isolation, or in vivo transplantation assays, can be performed with sorted side population cells to address questions regarding the functionality and the gene regulation of the side population cells. After watching this video, you should have a good understanding of how to isolate the side population of cells from zebrafish T-cell acute lymphoblastic leukemia.

isolation-side-population-myc-induced-t-cell-acute-lymphoblastic

Research

JoVE Journal

Immunology and Infection

6.5K visualizações.  The University of Chicago. The overall goal of this procedure is to isolate side population cells from a zebrafish model of myc-induced T-cell acute lymmphoblastic leukemia. The side population assay has been used to identify stem and progenitor cell populations in many normal and cancer tissues. But it hasn't yet been applied to any zebrafish cancer models.The main advantage of this technique is that it allows the prospective isolation of a population of cells in zebrafish T-ALL that may be enriched for cancer ...