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>

<ol>
	<li>Paik, E. J., 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=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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptome+analysis+of+fish+immune+cells+provides+insight+into+the+evolution+of+vertebrate+immune+cell+types.">Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types.</a> Genome Research. 27 (3), 451-461 (2017).</li><li>Tang, Q., 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=Dissecting+hematopoietic+and+renal+cell+heterogeneity+in+adult+zebrafish+at+single-cell+resolution+using+RNA+sequencing.">Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.</a> Journal of Experimental Medicine. 214 (10), 2875-2887 (2017).</li></ol>

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...

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 &#181;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>lck:eGFP</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>rag2:hMYC-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<...

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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6.9K 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 ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...