Name: isolation of the side population in myc-induced t-cell acute lymphoblastic leukemia in zebrafish
Uploaded: 2017-05-04T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish at JoVE.com

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:green fluorescent protein (rag2:GFP) plasmid DNA containing inverted terminal repeats recognized by tol2 transposase (see the Table of Materials for source of plasmid DNA)12 and elute the DNA in sterile water. 
		NOTE: GFP is used in this study, but any fluorescent protein will work.</li>
		<li>Prepare pCS2-transposase (tol2) RNA
		<ol>
			<li>Isolate tol2 plasmid DNA using commercially available kits (see the Table of Materials for the source of plasmid DNA)12 and elute the DNA in sterile water.</li>
			<li>Incubate 1 &#181;g of tol2 DNA at 37 &#176;C for 1 h with 5 units of NotI restriction enzyme in the appropriate buffer to linearize the plasmid. Heat-inactivate the NotI at 65 &#176;C for 20 min.</li>
			<li>Transcribe the tol2 RNA with SP6 RNA polymerase from the linearized DNA using a commercially available RNA transcription kit13. Store the RNA in single-use aliquots at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Prepare the injection mix on ice by adding 250 ng of rag2:c-myc plasmid DNA, 250 ng of rag2:GFP plasmid DNA, 150 ng of tol2 RNA, and 0.5 &#181;L of phenol red. Adjust the volume to 10 &#181;L with nuclease-free water.</li>
		<li>Inject ~1 nL of injection mix into the cell of a one-cell stage syngeneic zebrafish embryo using a microinjector and needle pulled from a glass capillary tube, following previously established methods8,14. 
		NOTE: Syngeneic zebrafish generated by parthenogenesis15 are used here to eliminate genetic heterogeneity and immune rejection after transplantation. However, wildtype zebrafish or any desired strain can also be used.</li>
		<li>Remove dead, underdeveloped, or malformed embryos at 24 h and 48 h post-injection16.</li>
		<li>Place the injected fish on the fish system at 6 days post-injection and raise them according to standard protocol6.</li>
	</ol>
	</li>
	<li>Screening injected zebrafish and monitoring tumor growth
	<ol>
		<li>At 21 days post-injection, place a single injected larva in a droplet of embryo medium on a Petri dish and screen for GFP fluorescence using an epifluorescent microscope (excitation bandpass: 470/40, emission bandpass: 525/50)8. 
		NOTE: Zebrafish larvae that have integrated both rag2:c-myc and rag2:GFP will have a green fluorescent thymus (positive fish) and may already exhibit the expansion of malignant green fluorescent cells beyond the thymus. All fish with a GFP+ thymus will go on to develop a GFP+ T-ALL. The larvae that do not demonstrate integration of the plasmids will not have any green fluorescent cells (negative fish).</li>
		<li>Separate the negative fish (based on a lack of fluorescence) into a different tank and monitor the fish again at 28 and 35 days post-injection. At 28 and 35 days post-injection, anesthetize the fish with 0.02% buffered tricaine (MS-222) in fish system water prior to screening.</li>
		<li>Place each zebrafish larva that is positive for a fluorescently labeled thymus or a fluorescently labeled tumor in an individual tank for monitoring tumor growth three times a week.</li>
	</ol>
	</li>
	<li>Collection of fluorescently labeled T-ALL cells from the primary tumor fish
	<ol>
		<li>Prepare at least 20 mL of 10% heat-inactivated fetal bovine serum in 0.9x phosphate buffered saline (FBS/PBS).</li>
		<li>Add 1 mL of FBS/PBS to one vial of heparin sodium salt (300 units).</li>
		<li>Select fish bearing tumors in more than 50% of the body, or those fish having difficulty eating or swimming. Sacrifice the fish by overdosing them with tricaine or by ice water immersion. Confirm death by lack a of gill movement and heartbeat.</li>
		<li>Use a razor blade to remove the head of the fish.</li>
		<li>Wash the cavity of the fish with the heparin solution to remove excess red blood cells.
		<ol>
			<li>Using a pipette, inject 100 &#181;L of heparin solution (prepared in step 1.3.2) into the body cavity of the fish. Remove all liquid that spills out (it will contain blood cells) and pipette it into a 1.5 mL microcentrifuge tube.</li>
			<li>Use a fresh pipette tip to wash the body again. Perform at least three washes, or until the liquid that spills out appears free of blood.</li>
			<li>Discard all liquid from the washes in steps 1.3.5.1 and 1.3.5.2, as it will not be used in further analyses.</li>
		</ol>
		</li>
		<li>Collect the leukemia cells.
		<ol>
			<li>Inject 100 &#181;L of FBS/PBS into the body cavity of the fish.</li>
			<li>Apply gentle pressure to the outside of the body of the fish with the pipette tip to squeeze/push out the tumor cells. 
			NOTE: If this step is performed using a dissecting microscope, the tumor cells spilling out of the body cavity will be evident.</li>
			<li>Collect the liquid and cells from each wash into a 1.5 mL microcentrifuge tube.</li>
			<li>Repeat steps 1.3.6.1-1.3.6.3 until most of the tumor cells are collected (usually 5-10 times). Collect all tumor cells into the same 1.5-mL microcentrifuge tube. Keep the cells at room temperature.</li>
		</ol>
		</li>
		<li>Mix the collected tumor cells from step 1.3.6 by gently pipetting them to dissociate the clumps of cells. Filter the cell suspension through a 40-&#181;m mesh filter. Wash the filter 1-2 times with 100 &#181;L of FBS/PBS. Keep the cells at room temperature.</li>
		<li>Mix 2 &#181;L of cell suspension with 18 &#181;L of trypan blue (0.4%, diluted 1:10 and filtered) in a new tube. Load 10 &#181;L on a hemocytometer and count the number of live (non-blue) fluorescent cells to calculate the number of isolated tumor cells. 
		NOTE: Non-blue GFP-negative cells are likely normal, non-malignant T-cells and should not be counted. At least 2 x 106 cells are required to stain the cells with Hoechst 33342.</li>
	</ol>
	</li>
</ol>
2. Staining the Fluorescently Labeled T-ALL Cells with Hoechst 33342 to Identify the Side Population
<ol>
	<li>Dilute Hoechst 33342 1:10 with nuclease-free H2O to make the 1 mg/mL working concentration. Store the dye at 4 &#176;C and in the dark.</li>
	<li>Prepare a 100-mM solution of verapamil by dissolving 49.1 mg in 1 mL of dimethyl sulfoxide (DMSO). 
	NOTE: Verapamil is an inhibitor of ABC transporters and is an important control for side population experiments. Adding verapamil to samples will inhibit the ability of the ABC transporters to efflux the Hoechst 33342 dye. Therefore, a true side population will disappear when verapamil is added to the sample.</li>
	<li>Set a water bath to 28 &#176;C.</li>
	<li>Prepare samples and controls in the dark by adding cells and solutions to fluorescence-activated cell sorter (FACS) tubes.
	<ol>
		<li>Prepare the samples by adding 106 cells, 15 &#181;L of 1 mg/mL Hoechst 33342, and 2.5 &#181;L of DMSO to a FACS tube. Adjust the volume to 1 mL with FBS/PBS.</li>
		<li>Prepare the controls by adding 106 cells, 15 &#181;L of 1 mg/mL Hoechst 33342, and 2.5 &#181;L of 100 mM verapamil to a FACS tube. Adjust the volume to 1 mL with FBS/PBS. 
		NOTE: A minimum of 106 cells/sample is recommended, but staining more cells is preferred, especially if the side population cells will be sorted for further analysis. Do not exceed 106 cells/mL, and maintain the Hoechst 33342 concentration at 15 &#181;g/mL. If 10 x 106 cells are stained, the total reaction volume is 10 mL.</li>
	</ol>
	</li>
	<li>Incubate in a 28 &#176;C water bath in the dark for 120 min.</li>
	<li>After the incubation, place the cells immediately on ice and keep them in the dark.</li>
	<li>Pellet the cells at 4 &#176;C for 6 min at 300 x g. Remove the supernatant. Wash with 1 mL of FBS/PBS.</li>
	<li>Pellet the cells at 4 &#176;C for 6 min at 300 x g. Remove the supernatant. Resuspend the cells in 1 mL of FBS/PBS.</li>
	<li>Keep at 4 &#176;C until cell sorting.</li>
	<li>15 min prior to cell sorting, add 2 &#181;L of propidium iodide (PI) stock (1 mg/mL) to each 1 mL of FBS/PBS (final concentration: 2 &#181;g/mL). Mix well.</li>
</ol>
3. Use FACS to Find the Side Population within the Fluorescently Labeled T-ALL Cell Population
<ol>
	<li>Analyze the stained zebrafish T-ALL cell suspensions on a cell sorter equipped with a UV laser for Hoechst 33342 dye excitation and a 488-nm laser for PI excitation and for the detection of the GFP+ T-ALL cells.</li>
	<li>Determine a gate for the live, GFP+ T-ALL cells.
	<ol>
		<li>Use forward scatter (FSC) and side scatter (SSC) area to gate out debris. 
		NOTE: FSC is used to distinguish cell size and SSC is used to distinguish the granularity of the cell.</li>
		<li>Use FSC and SSC width to discriminate doublets. 
		NOTE: There are three components identified by the flow cytometer detectors: area, height, and width. Area is typically used to measure the fluorescence of the cell. Width measures the time of flight and is twice as wide if two cells are clumped together.</li>
		<li>For live tumor cells, draw a gate around the cells positive for GFP and negative for PI using the appropriate detectors (e.g., FITC for GFP and PerCP Cy5-5 for PI).</li>
	</ol>
	</li>
	<li>In the live cell gate, measure the Hoechst 33342 fluorescence through the Hoechst Blue and Hoechst Red bandpass filters (450/50 and 670/30, respectively). 
	NOTE: The side population, if present, will be evident after this is set up. Importantly, the side population must disappear in the verapamil control in order for it to be considered a true side population.</li>
	<li>If sorting the side population cells for further experiments, use the large nozzle tip (100 &#181;m) and collect the cells into a FACS tube containing 3 mL of 100% FBS. Use a larger nozzle tip (100 &#181;m) instead of the smaller tip (70 &#181;m) to improve cell viability after sorting.</li>
</ol>

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The authors have nothing to disclose.

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 colleagues (2008) dissected zebrafish kidney marrow for the side population assay, the incubations were carried out at 25 &#176;C5. This incubation temperature was not sufficient for zebrafish T-ALL cells, as it resulted in a weak Hoechst 33342 profile (data not shown). In the protocol described here, an incubation temperature of 28 &#176;C was optimal to maintain cell viability and to achieve a strong Hoechst 33342 profile.
A second condition for the side population assay that requires consideration and prior testing is the concentration of Hoechst 33342. If the concentration of Hoechst 33342 is too low, the side population may not be visible, or the side population phenotype (dye efflux capability) may be falsely increased, identifying more side population cells than are actually present2. On the other hand, too high of a concentration of Hoechst 33342 can be toxic to the cells. For zebrafish T-ALL cells, 15 &#181;g/mL Hoechst 33342 incubated for 120 min is the optimal combination. Lower concentrations of Hoechst 33342 were tried for zebrafish T-ALL cells, but the side population was not always evident (data not shown). A total incubation time of 90 min was sufficient to detect a side population, but the maximal number of side population cells was seen after 120 min of incubation time.
As reported previously for mammalian systems, not every tumor will have a detectable side population17,18,19. Using this method, we observed a side population in 14 of 17 T-ALLs tested (data not shown). Having isolated side population cells from T-ALLs, this method is expected to be applicable to other zebrafish tumor models.
Within the heterogeneous zebrafish T-ALL tumor, leukemia initiating cells (LICs) are capable of reinitiating tumor growth and as such are believed responsible for cancer recurrence and metastasis. Currently, there is no method in zebrafish that allows for the prospective isolation of LICs in vivo. Calculating the LIC frequency requires tumor cell transplantation at a limiting dilution8,10. Previously published studies with clonal zebrafish have identified that LICs make up 0.1-1.4% of primary T-ALL cells10. Here, similar variability in the frequency of side population cells (0.2-1.0%) from clonal zebrafish T-ALLs is reported (Figure 2). Further experiments are needed to determine whether the side population is enriched in LICs; however, the similarity between the ranges of LIC frequency and the percentage of side population cells in these T-ALLs supports this hypothesis.
While the side population assay is sensitive and not always easy to perform, the assay allows researchers, for the first time, to isolate a rare population of cells in zebrafish T-ALL that may have stem or progenitor cell properties. There are currently no cell surface markers available in zebrafish to identify stem- or progenitor-like cells; hence, the ability to isolate the side population is very promising. Future applications of this method include limit dilution transplantation experiments of sorted side population T-ALL cells to assess the enrichment of LICs. In addition, this method will allow characteristics that define LICs to be elucidated, including the molecular mechanisms that govern LICs in zebrafish T-ALL. These experiments could provide starting points for the discovery of new therapeutic targets for T-ALL.

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><tbody><tr><td>Syngeneic zebrafish (CG2)</td><td></td><td></td><td>See Mizgireuv &lt;em&gt;et al.&lt;/em&gt;, 2006</td></tr><tr><td>&lt;em&gt;rag2&lt;/em&gt;:c-myc plasmid</td><td></td><td></td><td>Langenau Laboratory</td></tr><tr><td>&lt;em&gt;rag2&lt;/em&gt;:GFP plasmid constructed in de Jong Lab</td><td></td><td></td><td>&lt;em&gt;rag2&lt;/em&gt; promoter from Langenau Laboratory</td></tr><tr><td>pCS2-transposase plasmid</td><td></td><td></td><td>Chien Laboratory</td></tr><tr><td>NotI -HF restriction enzyme</td><td>New England Bio-Labs Inc.</td><td>R3189S</td><td>500 units in 25 &amp;micro;L</td></tr><tr><td>mMessage Machine SP6 Transcription Kit</td><td>Ambion</td><td>Thermo Fisher Scientific - AM1340</td><td>25 reactions</td></tr><tr><td>QIAGEN Plasmid Midi Kit</td><td>Qiagen, Inc.</td><td>12643</td><td></td></tr><tr><td>SteREO Discovery V8 Microscope</td><td>Carl Zeiss Microscopy</td><td>495015-0008-000</td><td></td></tr><tr><td>Digital microinjector</td><td>Tritech Research</td><td>MINJ-D</td><td></td></tr><tr><td>Brass straight-arm needle holder</td><td>Tritech Research</td><td>MINJ-4</td><td></td></tr><tr><td>Magnetic base and stand</td><td>Tritech Research</td><td>MINJ-HBMB</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige International</td><td>MN153</td><td></td></tr><tr><td>Glass capillaries</td><td>Sutter Instrument Co.</td><td>B100-50-10</td><td>10 cm without filament</td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf North America Inc.</td><td>930001007</td><td></td></tr><tr><td>Phenol Red solution</td><td>Sigma Life Science</td><td>P0290</td><td>0.5% concentration 100 mL</td></tr><tr><td>Plastic Transfer pipettes</td><td>Fisherbrand</td><td>137119D</td><td>graduated 7.5 mL bulb</td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Western Chemical, Inc.</td><td>Fisher Scientific - NC0342409</td><td>100 g</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HyClone Laboratories, Inc.</td><td>Fisher Scientific - SH3007103</td><td>500 mL</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco by Life Technologies</td><td>1001-023</td><td>pH 7.4 (1x) - 500 mL</td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>2106</td><td>15 - 300 unit vials</td></tr><tr><td>40 &amp;micro;m mesh filter</td><td>Falcon Corning Brand</td><td>352340</td><td>Case of 50</td></tr><tr><td>Trypan blue</td><td>Sigma Life Science</td><td>T8154</td><td>20 mL - Solution (0.4%)</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td>10 mL</td></tr><tr><td>Verapamil</td><td>Sigma Life Science</td><td>V4629</td><td>1 g</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Life Science</td><td>D8418</td><td>100 mL</td></tr><tr><td>Propidium Iodide</td><td>Life Technologies</td><td>P3566</td><td>10 mL</td></tr><tr><td>FACSAria Fusion cell sorter</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva 8.0.1</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo v10.2</td><td>FlowJo, LLC</td><td></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).
In the representative example, a fish with a GFP+ T-ALL was stained with Hoechst 33342 to determine the percentage of cells in the side population. Figure 1 shows the gating scheme used to determine the percentage of side population cells. First, using FSC and SSC, the T-ALL cells are isolated by excluding debris (Figure 1A) and subsequently selecting against doublets (Figure 1B). Next, all live cells are isolated by gating cells that are GFP+ and negative for the dead cell discriminator PI (Figure 1C). Once the single, live T-ALL cells are selected, the side population can be found in the Hoechst 33342 profile. In the main cell population, Hoechst 33342 remains in the cells, and these cells can be separated into groups by DNA content. However, the side population of cells are distinguishable from the main cell population, as the side population cells efflux the dye due to higher numbers of ABC transporters on the cell membrane. To visualize the side population, the Hoechst profile is used to compare two channels, Hoechst Blue and Hoechst Red. The side population is the dim tail of cells extending from the left side of the main cell population towards the Hoechst Blue axis (Figure 1D).
A true side population will be lost when the cells are treated with an inhibitor of ABC transporters, such as verapamil. Figure 2 shows the side populations in several representative T-ALL tumors, as well as when those tumors are treated with verapamil during the staining with Hoechst 33342. To ensure that the population found in the sample is a true side population, those cells must disappear in the presence of the inhibitor, as is shown in Figure 2.
<table height="525" width="487">
	<tbody>
		<tr>
			<td>Stock Number</td>
			<td>Number Fish Screened</td>
			<td>Number Positive</td>
			<td>Rate of Transgenesis</td>
		</tr>
		<tr>
			<td>930</td>
			<td>21</td>
			<td>3</td>
			<td>14.3%</td>
		</tr>
		<tr>
			<td>934</td>
			<td>12</td>
			<td>4</td>
			<td>33.3%</td>
		</tr>
		<tr>
			<td>950</td>
			<td>12</td>
			<td>5</td>
			<td>41.7%</td>
		</tr>
		<tr>
			<td>951</td>
			<td>19</td>
			<td>3</td>
			<td>15.8%</td>
		</tr>
		<tr>
			<td>960</td>
			<td>3</td>
			<td>1</td>
			<td>33.3%</td>
		</tr>
		<tr>
			<td>983</td>
			<td>9</td>
			<td>4</td>
			<td>44.4%</td>
		</tr>
		<tr>
			<td>1004</td>
			<td>30</td>
			<td>3</td>
			<td>10.0%</td>
		</tr>
		<tr>
			<td>1013</td>
			<td>4</td>
			<td>1</td>
			<td>25.0%</td>
		</tr>
		<tr>
			<td>1025</td>
			<td>35</td>
			<td>5</td>
			<td>14.3%</td>
		</tr>
		<tr>
			<td>1029</td>
			<td>10</td>
			<td>2</td>
			<td>20.0%</td>
		</tr>
		<tr>
			<td>1065</td>
			<td>5</td>
			<td>1</td>
			<td>20.0%</td>
		</tr>
		<tr>
			<td>1070</td>
			<td>6</td>
			<td>1</td>
			<td>16.7%</td>
		</tr>
		<tr>
			<td>1073</td>
			<td>19</td>
			<td>8</td>
			<td>42.1%</td>
		</tr>
		<tr>
			<td>1079</td>
			<td>8</td>
			<td>1</td>
			<td>12.5%</td>
		</tr>
		<tr>
			<td>1105</td>
			<td>38</td>
			<td>7</td>
			<td>18.4%</td>
		</tr>
		<tr>
			<td>1193</td>
			<td>30</td>
			<td>8</td>
			<td>26.7%</td>
		</tr>
		<tr>
			<td>1215</td>
			<td>9</td>
			<td>1</td>
			<td>11.1%</td>
		</tr>
		<tr>
			<td>1229</td>
			<td>10</td>
			<td>2</td>
			<td>20.0%</td>
		</tr>
		<tr>
			<td>1314</td>
			<td>6</td>
			<td>1</td>
			<td>16.7%</td>
		</tr>
	</tbody>
</table>
Table 1: Transgenesis Rates When Using tol2 Transposase for Plasmid Microinjection into Zebrafish Embryos. The calculated rates of transgenesis are shown for nineteen separate injection days. The Number of Fish Screened represents the number of fish that remained alive 21 days post-injection, and the Number Positive represents the number of fish that were positive for fluorescent tumors.
<img alt="Figure 1" src="/files/ftp_upload/55711/55711fig1.jpg" /> 
Figure 1: Side Population Gating Scheme. An example of the gating scheme used to identify the side population in a GFP+ T-ALL in zebrafish. (A) First, the debris is excluded using forward scatter (FSC) and side scatter (SSC) area. (B) Next, single cells are isolated by discriminating against doublets using FSC and SSC width parameters. (C) Since this example tumor was GFP+, all live cells must be GFP+ and negative for the dead cell marker Propidium Iodide. (D) The side population can be seen with the Hoechst Red versus Hoechst Blue profile. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55711/55711fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55711/55711fig2.jpg" /> 
Figure 2: Side Population Disappears When Treated with the ABC Transporter Inhibitor, Verapamil. Gates for the side population are shown. The top panels show four different tumor samples. It is important to note that not all tumors have the same side population percentage, and the side population can look different for each tumor tested. The bottom panels show tumor samples that were treated with verapamil during the staining with Hoechst 33342 to block dye efflux. While the side populations may look different for each tumor, the effect of the verapamil is the same: the side population disappears. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55711/55711fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

6.5K Views.  The University of Chicago. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Developmental Biology

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Estimating LSC Frequency in Zebrafish T-ALL Using Cell Proliferation Dye Sorting

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Author Spotlight: Identification and Isolation of Quiescent Leukemia Stem Cells from Zebrafish T-ALL

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

Cancer Research

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Biology

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

Medicine

Fluorescently Labeled T-ALL Cells Isolation: A Method to Isolate T-ALL Cells from Adult Zebrafish

Source: Pruitt, M. M., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/55711">Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish.</a> J. Vis. Exp. (2017).
This protocol describes the technique of isolating fluorescently labeled T-cell acute lymphoblastic leukemia (T-ALL) tumor cells from an adult zebrafish and quantifying them using trypan blue dye.

Fluorescencyjnie znakowana izolacja komórek T-ALL: metoda izolacji komórek T-ALL od dorosłych danio pręgowanego

Source: Pruitt, M. M., et al. Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish. J. Vis. Exp. (2017).
This ...

JoVE Encyclopedia of Experiments

Leukemia

Cell isolation & characterization

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

Source: Pruitt, M. M., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/55711">Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish.</a> J. Vis. Exp. (2017).
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.

Test populacji bocznej: metoda izolowania bocznej populacji komórek nowotworowych od danio pręgowanego

Source: Pruitt, M. M., et al. Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish. J. Vis. Exp. (2017).
The ...

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

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Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Fluorescencyjnie znakowana izolacja komórek T-ALL: metoda izolacji komórek T-ALL od dorosłych danio pręgowanego

Test populacji bocznej: metoda izolowania bocznej populacji komórek nowotworowych od danio pręgowanego