We would like to thank Megan Malone-Perez for zebrafish care, and the OUHSC flow cytometry core. This work was supported&#160;by grants from Hyundai Hope on Wheels, the Oklahoma Center for the Advancement of Science and Technology (HRP-067), an NIH/NIGMS INBRE pilot project award (P20 GM103447). JKF holds the E.L. &#38; Thelma Gaylord Endowed Chair in Pediatric Hematology-Oncology of the Children&#39;s Hospital Foundation.

All procedures involving zebrafish were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Oklahoma Health Sciences Center.
1. Isolating Non-malignant B and T Lymphocytes from Transgenic lck:eGFP Fish
<ol>
	<li>Anesthetize the fish using 0.02% tricaine (MS-222) in fish system water.</li>
	<li>Examine 2&#8211;6 month old fish for fluorescent thymi, which are located at the dorsomedial aspect of the branchial cavity of zebrafish and other teleosts14. Use an epifluorescence microscope (470/40 excitation wavelength and 525/50 emission filter) to detect GFP.</li>
	<li>Prepare in advance 50 mL of filter-sterilized 1x Roswell Park Memorial Institute Medium (RPMI) containing 1% fetal bovine serum and 1% penicillin-streptomycin (sorting media). Unused sorting media can be stored at 4 &#176;C for up to 2 months.</li>
	<li>Euthanize the fish by placing it in a beaker containing 0.2% Tricaine for approximately 5 min, followed by ice bath immersion. Confirm death by the cessation of opercular (gill) movement.</li>
	<li>Place the euthanized fish in a Petri dish and dissect lymphoid organs of interest, using the fluorescent microscope to dissect the GFP+ thymi14, and bright field microscopy settings to dissect the kidney marrow and spleen15. Results presented here were obtained using 3 month-old fish. Lymphocyte proportions and absolute numbers vary by age and genotype (see discussion for further detail).</li>
	<li>Place each dissected organ in a 1.5 mL tube containing 500 &#181;L of cold sorting media.</li>
	<li>Homogenize the tissue on ice using a pestle micro-tube homogenizer.</li>
	<li>Pass homogenized tissue through a 35 &#181;m mesh filter to generate a single cell suspension. Keep the cells on ice until analysis.</li>
</ol>
2. Isolating Malignant Lymphocytes from Double-transgenic rag2:hMYC;lck:eGFP Zebrafish
<ol>
	<li>Beginning at 2-4 months, microscopically screen rag2:hMYC; lck:eGFP fish for abnormal GFP patterns. 
	NOTE: B cells are GFPlo in the lck:eGFP background, so B-ALL requires practice to recognize; T-ALL is obvious, because T cells are GFPhi. Consequently, the rag2:hMYC, lck:eGFP dual-transgenic line has two phenotypes: brightly-fluorescent T-ALL, which usually arise from the thymus and extend into the body, and dimly-fluorescent B-ALL.
	<ol>
		<li>Using a fluorescent microscope, screen the fish using &#8220;low exposure&#8221; settings (200 ms, 2.4x gain) to identify bright T-ALL (Figure 1A) and &#8220;high exposure&#8221; (1.5 s, 3.4x gain) to identify dim B-ALL (Figure 1A). WT controls and pre-leukemic fish have GFP localized only in the thymus (Figure 1B).</li>
	</ol>
	</li>
	<li>Anesthetize and examine the fish as described in steps 1.1&#8211;1.2.</li>
	<li>Categorize the fish based on the extent of GFP fluorescence, using a simple three category system: 
	NOTE: Level 1: Fluorescence appears as a thymic tumor with only limited local spread. 
	Level 2: Fluorescence appears beyond the thymus, involving &#60;50% of the body. 
	Level 3: Fluorescence extends beyond 50% of the body.</li>
	<li>Separate the fish with ALL from those without cancer. Monitor pre-leukemic fish (i.e., fish without GFP+ tumors) once monthly for the development of new ALL. By ~9 months, all rag2:hMYC, lck:eGFP fish develop T-ALL, B-ALL, or both.</li>
	<li>For T- or B-ALL cell isolation, select Level 3 fish (ALL involving &#62;50% of the body), which yield more than 2 x 106 ALL cells, and typically many more.</li>
	<li>Euthanize the fish as in step 1.4. 
	NOTE: There are two methods to obtain ALL cells: whole body homogenization and a peritoneal wash technique16. The methods differ in the absolute number of cells collected for sorting, and thus, the amounts of FACS time required and cost (see discussion for further detail).</li>
	<li>For either method, first place the euthanized fish in a Petri dish and using a razor blade, remove the head including the thymic region. This can be processed separately, if desired, or used for histological staining.</li>
	<li>Peritoneal Wash Method
	<ol>
		<li>Using a P1000 pipette, wash the fish peritoneal cavity with 500 &#181;L of cold sorting media, collecting the cells and media in a 5 mL tube.</li>
		<li>Using a fresh pipette tip, inject an additional 200&#8211;300 &#181;L of cold sorting media into the body cavity. Then, using the tip of the pipette, apply gentle pressure to the fish body to extrude the cells out of the body cavity. Collect this media and add to the 5 mL tube.</li>
		<li>Repeat step 2.8.2 2&#8211;3 times. Keep the collected cells in sorting media on ice.</li>
		<li>Filter the cell suspension though a 35 &#181;m mesh filter prior to flow cytometry/FACS and keep the cells on ice until analysis.</li>
	</ol>
	</li>
	<li>Whole Body Homogenization
	<ol>
		<li>After removing the fish head, place the body in a 1.5 mL tube containing 200 &#181;L of sorting media.</li>
		<li>Homogenize the body using a pestle micro-tube homogenizer.</li>
		<li>Add an additional 300 &#181;L of cold sorting media. Filter the cell suspension though a 35 &#181;m mesh filter. Add sorting media as needed to wash all cells through the filter, until only tissue debris remains on the filter. Keep the cells on ice until analysis.</li>
	</ol>
	</li>
</ol>
3. Cytometric Analysis of Normal or Malignant B Lymphocytes
<ol>
	<li>Set flow cytometric analysis and/or FACS parameters according to manufacturer&#8217;s guide.</li>
	<li>Acquire desired number of events to initially characterize the sample. Analyze 1 x 104 to 5 x 104 events prior to sorting to determining specific gates for subsequent sorting steps.
	<ol>
		<li>Determine gates: Define lymphocyte and progenitor cell gates using forward scatter (FSC) and side scatter (SSC) parameters, excluding cellular debris (Figure 2A-C)17. FSC and SSC correspond to cell size/diameter and granularity, respectively. 
		NOTE: GFP-, GFPlo, and GFPhi cells differ 10-to-100-fold in terms of their GFP fluorescence intensity, making separation of these populations straightforward (Figures 2-5). Live-dead discrimination can be assessed at this point using propidium iodine (PI) or 7-aminoactinomycin D (7-AAAD) viability staining. Previous experiments with PI typically demonstrate &#62;95% viability of GFP+ cells after FACS.</li>
		<li>Exclude the cell doublets, according to the parameters of the FACS machine being used.</li>
		<li>Within the lymphoid and/or precursor gates, determine the number and percentage of GFP+ cells.</li>
	</ol>
	</li>
	<li>Use phycoerythrin (PE) and GFP intensities, define gate for GFP- vs. GFPlo vs. GFPhi cells. 
	NOTE: B cells exhibit dim GFP fluorescence and T cells show bright GFP in lck:eGFP lines. Many lymphoid organs or tumor samples contain both GFP+ populations. B-lineage cells/B-ALL are GFPlo and T-lineage cells/T-ALL are GFPhi. Define gates to distinguish between GFP-, GFPlo, and GFPhi cells, and collect these populations separately (Figure 2A-C, Figure 3A-C, Figure 4A and Figure 5A).</li>
	<li>Sort each cell population into different 15 mL polypropylene tubes containing 2 mL of sorting media, or directly into different 1.5 mL tubes containing an appropriate buffer for further analyses (e.g., RNA, DNA, or protein extraction, allo-transplantation, etc.).</li>
	<li>Keep purified cells on ice prior to further analyses.</li>
</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=Hematopoietic+development+in+the+zebrafish.">Hematopoietic development in the zebrafish.</a> International Journal of Developmental Biology. 54 (6-7), 1127-1137 (2010).</li><li>Kasheta, 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=Identification+and+characterization+of+T+reg-like+cells+in+zebrafish.">Identification and characterization of T reg-like cells in zebrafish.</a> Journal of Experimental Medicine. 214 (12), 3519-3530 (2017).</li><li>Liu, X., 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=Zebrafish+B+Cell+Development+without+a+Pre-B+Cell+Stage,+Revealed+by+CD79+Fluorescence+Reporter+Transgenes.">Zebrafish B Cell Development without a Pre-B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes.</a> Journal of Immunology. 199 (5), 1706-1715 (2017).</li><li>Page, 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=An+evolutionarily+conserved+program+of+B-cell+development+and+activation+in+zebrafish.">An evolutionarily conserved program of B-cell development and activation in zebrafish.</a> Blood. 122 (8), e1-e11 (2013).</li><li>Schorpp, 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=Conserved+functions+of+Ikaros+in+vertebrate+lymphocyte+development:+genetic+evidence+for+distinct+larval+and+adult+phases+of+T+cell+development+and+two+lineages+of+B+cells+in+zebrafish.">Conserved functions of Ikaros in vertebrate lymphocyte development: genetic evidence for distinct larval and adult phases of T cell development and two lineages of B cells in zebrafish.</a> Journal of Immunology. 177 (4), 2463-2476 (2006).</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=In+vivo+tracking+of+T+cell+development,+ablation,+and+engraftment+in+transgenic+zebrafish.">In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish.</a> Proceedings of National Academy of Sciences U S A. 101 (19), 7369-7374 (2004).</li><li>Trede, N. S., Langenau, D. M., Traver, D., Look, A. T., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+to+understand+immunity.">The use of zebrafish to understand immunity.</a> Immunity. 20 (4), 367-379 (2004).</li><li>Frazer, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+T-cell+malignancy+models+established+in+a+zebrafish+phenotypic+screen.">Heritable T-cell malignancy models established in a zebrafish phenotypic screen.</a> Leukemia. 23 (10), 1825-1835 (2009).</li><li>Rudner, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+acquired+genomic+changes+in+zebrafish+and+human+T-ALL.">Shared acquired genomic changes in zebrafish and human T-ALL.</a> Oncogene. 30 (41), 4289-4296 (2011).</li><li>Gutierrez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pten+mediates+Myc+oncogene+dependence+in+a+conditional+zebrafish+model+of+T+cell+acute+lymphoblastic+leukemia.">Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia.</a> Journal of Experimental Medicine. 208 (8), 1595-1603 (2011).</li><li>Borga, 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=Simultaneous+B+and+T+cell+acute+lymphoblastic+leukemias+in+zebrafish+driven+by+transgenic+MYC:+implications+for+oncogenesis+and+lymphopoiesis.">Simultaneous B and T cell acute lymphoblastic leukemias in zebrafish driven by transgenic MYC: implications for oncogenesis and lymphopoiesis.</a> Leukemia. , (2018).</li><li>Haferlach, 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=Clinical+utility+of+microarray-based+gene+expression+profiling+in+the+diagnosis+and+subclassification+of+leukemia:+report+from+the+International+Microarray+Innovations+in+Leukemia+Study+Group.">Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group.</a> Journal of Clinical Oncology. 28 (15), 2529-2537 (2010).</li><li>Novershtern, 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=Densely+interconnected+transcriptional+circuits+control+cell+states+in+human+hematopoiesis.">Densely interconnected transcriptional circuits control cell states in human hematopoiesis.</a> Cell. 144 (2), 296-309 (2011).</li><li>Menke, A. L., Spitsbergen, J. M., Wolterbeek, A. P., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+the+adult+zebrafish.">Normal anatomy and histology of the adult zebrafish.</a> Toxicology Pathology. 39 (5), 759-775 (2011).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. , (2010).</li><li>Pruitt, M. M., Marin, W., Waarts, M. R., de Jong, J. L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+the+Side+Population+in+Myc-induced+T-cell+Acute+Lymphoblastic+Leukemia+in+Zebrafish.">Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish.</a> Journal of Visualized Experiments. (37), (2017).</li><li>Traver, 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nature Immunology. 4 (12), 1238-1246 (2003).</li><li>Carmona, S. 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The authors declare no conflict of interests.

We developed and provide a protocol to isolate B cells from lck:eGFP transgenic zebrafish, adding this to other D. rerio models with B-lineage labels3,4. Somewhat surprisingly, the identification of GFPlo B cells in this line went unnoticed since its description in 2004.Generally, lck is considered to be T cell-specific6, but recent studies found unexpected lck expression by natural killer and myeloid cells, as well as in B cells as shown here18,19. In agreement with our discovery that zebrafish B cells are GFPlo, pre-B, na&#239;ve, and mature B cells in humans also express low levels of LCK11,13.
Due to the differing GFP levels in B and T cells of these fish, cells of both lineages can now be isolated from this line. Although these animals have classically been used to study thymic T lymphocyte development and tracking, we demonstrate that similar studies are also possible with B cells. To this end, we identify B cells in thymus, kidney marrow, and spleen here, and in peripheral blood and elsewhere in prior work11.
Recognizing B-ALL in rag2:hMYC; lck:eGFP fish requires a keen eye, but with practice, is straightforward. Next, choosing which method to use to purify ALL cells is critical. Here, we present two methods: peritoneal wash and whole body homogenization. Peritoneal washing yields a much lower number of total cells, but a very high percentage of GFP+ cells. Consequently, little FACS time is required, minimizing the cost. For downstream applications, where total yield is less important (e.g., qRT-PCR), this is more efficient. Alternatively, whole body homogenization results in larger single cell suspensions containing many more cells, but a lower percentage of cells will be GFP+. Thus, more FACS time is required to collect the same number of GFP+ cells as with peritoneal washing, but millions more cells can be purified. For downstream studies, where high yield is important (e.g., Western blot), this may offset the added cost. In addition, for ALL studies, total body homogenization captures the diversity of cancer cells present, more accurately representing tumor heterogeneity. Although not listed in our protocol, it is also feasible to dissect only GFP+ body regions/organs from fish with ALL for homogenization by mincing in a Petri dish, thus decreasing total FACS time and cost, akin to peritoneal washing.
Thymic GFP expression in lck:eGFP fish showed that GFP is detectable as early as 5 dpf6. Our studies here were performed in adult fish 3 months of age or older, and show that at this age, B cells are abundant in kidney marrow and spleen, but T cells are rare. Subsequent studies with fish of different ages will be required to determine if T cells are more prevalent at different time points. Likewise, at 3 months, T cells are enriched in the thymus, but a considerable number of thymic B cells are also present. If these lymphocyte populations vary at different ages is currently unknown, but overall, thymic fluorescence fades in lck:eGFP fish beginning at sexual maturity (~3 months), so it is likely that T cell numbers diminish with increasing age, and B cells may also. Likewise, when GFPlo B cells colonize the zebrafish thymus is currently unknown. Using lck:eGFP fish, it is now possible to monitor thymic B and T cell development, influx, efflux, and the specific kinetics of these events. In addition, such questions are also pertinent to other anatomic sites where B cells reside, such as kidney marrow, spleen, blood, and intestine (data not shown).
Besides B cell developmental studies, we recently found B-ALL in lck:eGFP; rag2:hMYC double-transgenic fish11. Transgenic rag2:hMYC was known to induce T-ALL10 but B-ALL had gone unrecognized. Due to the high penetrance of this oncogene, both types of ALL are prevalent in these fish, and many fish actually develop both ALL types simultaneously11. Since B-ALL are GFPlo, while T-ALL are GFPhi, varying GFP expression allows these two entities to be isolated by FACS, even when occurring in the same animal (Figure 5A). Crucially, in both normal and malignant lymphocytes, qRT-PCRresults demonstrate that GFPlo and GFPhi cells consistently correspond to the B- and T-lineages, respectively (Figure 2 and Figure 3).
The untapped potential of lck:eGFP fish was central to this work, highlighting the fact that many pre-existing transgenic lines likely have utility beyond their intended purpose. Here, we present a novel protocol to isolate B and T cells from D. rerio with transgenic lck:eGFP, opening the door to new investigational avenues concerning normal and malignant B lymphocytes. The results of such studies will undoubtedly re-vitalize what has already been a valuable resource to the hematopoiesis, lymphopoiesis, and cancer biology fields.

Zebrafish offer powerful attributes, such as genetic manipulability, high fecundity, optical translucency, and rapid development that facilitate studying vertebrate development using genetic approaches. These advantages, together with the shared features of teleost and mammalian hematopoiesis, make D. rerio ideal for in vivo analyses of lymphopoiesis and lymphocyte function, from their earliest appearance in larvae throughout adulthood. Blood development in zebrafish relies upon well-conserved genetic processes that are shared with mammals, and these extend to the adaptive immune system. Additionally, molecular mechanisms governing lymphoid development are remarkably conserved between zebrafish and mammals1.
Over the past 2 decades, transgenic D. rerio lines that label specific blood lineages and mutant lines deficient in these lineages have been created2,3,4,5. One of these, the lck:eGFP transgenic line, uses the zebrafish lymphocyte protein tyrosine kinase (lck) promoter to drive GFP expression6. This gene, which is highly expressed by both T-lineage precursors and mature T lymphocytes, allows in vivo tracking of thymic T cell development and ex vivo purification of T-lineage cells by FACS7. Previously, we used this line in a forward-genetic ENU mutagenesis screen to identify germline mutants prone to T-ALL and to study somatically-acquired genetic events linked to T cell oncogenesis8,9.
Recently, our laboratory further extended the utility of lck:eGFP zebrafish. In double-transgenic rag2:hMYC (human MYC), lck:eGFP D. rerio that are known to develop T-ALL 10, we discovered that B-lineage ALL also occur11. Unlike T-ALL in this model, which fluoresce brightly due to high GFP expression, B-ALL are dimly-fluorescent due to low GFP levels, allowing fish with B-ALL to be distinguished grossly from those with T-ALL by fluorescent microscopy. This differential GFP expression also permits the separation of GFPlo B-ALL cells from GFPhi T-ALL cells using FACS11. Moreover, low lck expression is not unique to zebrafish B-ALL, as human B-ALL also express low levels of LCK11,12. Likewise, normal B-lineage cells of D. rerio, mice, and humans also express low levels of lck/Lck/LCK, with immature B cells having the highest expression11,13. On a per cell basis, B-lineage cells in lckeGFP zebrafish or derivative lines express 1-10% as much GFP as T lymphocytes. These GFPlo cells express characteristic B cell mRNAs such as pax5, cd79b, blnk, btk, ighm, ighz, and others, and can be purified from marrow, thymus, spleen, or peripheral blood11. Therefore, both B- and T-lineage cells can be isolated from lckeGFP zebrafish, and in the case of rag2hMYC, lckeGFP animals, B- and T-ALL cells as well11.
Here, we present our protocol to efficiently FACS-purify non-malignant B cells from lck:eGFP zebrafish, and non-malignant or malignant B cells of rag2hMYC;lck:eGFP fish, using various source tissues. Such cells can likewise be quantified by flow cytometry without FACS isolation, if desired. Discovery of low lck expression-and consequently, low GFP expression-by B cells opens new doors of experimental possibilities for lckeGFP zebrafish, such as in vivo B cell developmental studies. Thus, this transgenic line, first reported in 2004, has new life as we seek to utilize it to glean fresh insights concerning zebrafish adaptive immunity.

<table><tbody><tr><td>35 &amp;micro;m mesh</td><td>Sefar Filter technology</td><td>7050-1220-000-13</td><td></td></tr><tr><td>5 mL Polystyrene round-Bottom tube with cell-strainer cap</td><td>Falcon Corning Brand</td><td>352235</td><td></td></tr><tr><td>50 mL conical tube</td><td>VWR international</td><td>525-0448</td><td></td></tr><tr><td>AZ APO 100 Fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Cytoflex</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>DS-Qi1MC camara</td><td>Nikon</td><td></td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methansesulfonate; MS-222</td><td>Sigma</td><td>E-10521</td><td></td></tr><tr><td>FACSJazz</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fetal bovine Serum</td><td>Thermo Fisher</td><td>10437028</td><td></td></tr><tr><td>FlowJo v10.2</td><td>FlowJo, LLC</td><td></td><td></td></tr><tr><td>&lt;em&gt;lck:eGFP&lt;/em&gt;</td><td></td><td></td><td>See Langeneu et al., 2004</td></tr><tr><td>NIS Elements software</td><td>Nikon</td><td>Version 4.13</td><td></td></tr><tr><td>Penicilin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Pestle micro-tube homogenizers</td><td>Electron Microscopy Sciences</td><td>64788-20</td><td></td></tr><tr><td>Plastic Transfer pippetes</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;rag2:hMYC&lt;/em&gt;-ER</td><td></td><td></td><td>See Gutierrez et al., 2011</td></tr><tr><td>RPMI Media 1640 1x</td><td>Life Technologies</td><td>11835-030</td><td></td></tr></tbody></table>

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.

We used flow cytometry to analyze and FACS to isolate GFPlo and GFPhi cells from thymus, kidney marrow, and spleen of lck:eGFP transgenic zebrafish. Analysis of 3-month-old fish revealed the thymus contained mostly GFP+ lymphocytes. GFP+ cells were largely confined to the lymphoid gate previously described by Traver et al.17. Two distinct GFP+ populations, GFPloand GFPhi,can be observed in the thymus. GFPhi lymphocytes represented a higher percentage, ~60%, while GFPlo cells were less abundant, representing ~40% of total thymic lymphocytes (Table 1). Unlike mammals, fish hematopoietic marrow is localized within the kidney, rather than within bone. We determined B cells residing in kidney marrow also express low levels of GFP (Figure 2B and Figure 3B). GFPlo cells in marrow were abundant, while GFPhi cells were scarce (Figure 2B and Figure 3B)), indicating that only a small percentage of T lymphocytes were present in the marrow at 3 months of age. Splenic samples likewise showed higher percentages of GFPlo than GFPhi cells (Figure 2C and Figure 3C). We also analyzed non-malignant lymphocytes from thymus, marrow, and spleen of double-transgenic lck:eGFP; rag2:hMYC zebrafish, which are prone to both B- and T-ALL11. In fish that had not yet developed ALL, results from thymus, marrow, and spleen were similar to single-transgenic lck:eGFP fish. However, the numbers of lymphocytes per organ were increased (Figure 3), presumably due to MYC-driven expansion of immature B and T cell populations where the rag2 promoter is active.
We also analyzed double-transgenic fish with B- and/or T-ALL that had developed fluorescent cancers by 6 months. Predictably, B-ALL fish with dimly-fluorescent cancers contained mostly GFPlo cells (Figure 4A). In contrast, brightly-fluorescent fish can harbor either isolated T-ALL or mixed populations of both GFPlo B-ALL and GFPhi T-ALL cells (Figure 5). An example of mixed ALL, which contains distinct B- and T-ALL, is shown here (Figure 5). To confirm the identities of GFPlo and GFPhi cells as B- and T-lineage, respectively, we analyzed B- and T-cell-specific transcripts by quantitative real-time PCR (qRT-PCR). Our results show GFPlo cells express higher levels of B cell transcripts and GFPhi cells express higher levels of T cell genes (Figure 2D, Figure3D, Figure4B and Figure5B). Furthermore, expression of lck and GFP in GFPlo ALL correspond to the dim in vivo GFP fluorescence of B-ALL (Figure 2D, Figure3D, Figure4B and Figure5B; qRT-PCR conditions and primers sequences previously described by Borga et al.)11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59191/59191fig1.jpg" /> 
Figure 1: Distinct fluorescence patterns in lck:eGFP transgenic fish. (A) Fluorescent microscopy images of rag2:hMYC; lck:eGFP fish with brightly-fluorescent T-ALL (left) or dimly-fluorescent B-ALL (right). T-ALL is visible with low exposure settings; B-ALL can only be seen using high exposures. (B) High exposure settings show faint thymic fluorescence (yellow circles) in wild-type lck:eGFP fish (left) and rag2:hMYC; lck:eGFP double-transgenic fish without ALL (right). Scale bars = 20 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59191/59191fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59191/59191fig2v2.jpg" /> 
Figure 2: Lymphocyte populations in lck:eGFP zebrafish. Images at top show a 3 month-old WT, lck:eGFP fish with high exposure settings or computer-enhancement (to facilitate visualization). Scale bars = 20 mm. Flow cytometric analyses of thymus (A), marrow (B), and spleen (C). Left panels show FSC (x-axis) and SSC (y-axis), with black ovals indicating lymphocyte gates. Middle panels depict fluorescence-based gating with GFP (x-axis) and PE (y-axis). GFPhi (blue rectangle), GFPlo (green rectangle) and GFP- (black ovals) populations are shown. Right panels display histograms of GFPhi and GFPlo cells. Dashed lines indicate gating criteria in middle panels. (D) WT lck:eGFP thymi sorted for GFPhi (blue) and GFPlo (green). Expression of B cell gene (pax5), T (cd4) cell-specific genes), lck and GFP. Results are normalized to housekeeping genes (&#946;-actin and eef1a1l1) and shown as fold-change &#177; Standard Error (S.E). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59191/59191fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59191/59191fig3v2.jpg" /> 
Figure 3: Lymphocyte populations in pre-leukemic rag2:hMYC; lck:eGFP zebrafish. Images at top show a 3-month-old rag2:hMYC; lck:eGFP fish with high and computer-enhanced exposures. Scale bar = 20 mm. Panels of parts A-D are depicted in identical format to Figure 2. (D) Expression of pax5, cd4, lck, and GFP in FACS-purified thymic GFPlo (green) GFPhi (blue) cell populations of lck:eGFP fish. qRT-PCR results are shown as fold-change &#177; S.E. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59191/59191fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59191/59191fig4.jpg" /> 
Figure 4: Analysis of B-ALL rag2:hMYC; lck:eGFP transgenic fish. (A)&#160;Top: 6-month-old rag2:hMYC; lck:eGFP fish with B-ALL using high exposure setting. Flow cytometric analyses of tumor cells isolated from fish body are identical format to Figure 2.&#160;(B)&#160;Gene expression in B-ALL GFPlo cells (green gate in Figure 4A), rag2:hMYC; lck:eGFP GFPhi thymocytes (blue gate in Figure 3A), and T-ALL GFPhi cells (blue gate in Figure 5A). Expression of B (pax5, cd79a) and T (cd4) cell-specific genes, lck, and GFP. Results are normalized to housekeeping genes (&#946;-actin and eef1a1l1) and shown as fold-change &#177; S.E. Scale bar = 20 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59191/59191fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59191/59191fig5.jpg" /> 
Figure 5: Analysis of mixed ALL rag2:hMYC; lck:eGFP transgenic fish. (A) Top: 6 month-old rag2:hMYC; lck:eGFP fish with mixed-ALL using high exposure setting. Flow cytometric analyses of tumor cells isolated from fish body are identical format to Figure 2. (B)&#160;GFPhi (blue) and GFPlo (green) FACS gates. Expression of B (pax5, cd79a) and T (cd4) cell-specific genes, lck, and GFP. Results are normalized to housekeeping genes (&#946;-actin and eef1a1l1) and shown as fold-change &#177; S.E. Scale bar = 20 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59191/59191fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish at JoVE.com

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.

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 ...

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7.0K Views.  University of Oklahoma Health Sciences Center. 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.

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

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

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

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Cancer Research

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Immunology and Infection

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 ...

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.

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 ...

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

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.

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).
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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.

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 ...

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

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Chapters in this video

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Isolation of Human Islets from Partially Pancreatectomized Patients

Isolation and Characterization of Single Cells from Zebrafish Embryos

Zebrafish Model of Neuroblastoma Metastasis

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

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

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

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

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

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