Name: isolating malignant and non-malignant b cells from lck:egfp zebrafish
Uploaded: 2019-02-22T15:00:00
Description: Watch this Scientific Journal Video about Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish at JoVE.com

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 a...

<ol>
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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>

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 ...

We recently found that many B cells in LCK GFP zebrafish express low levels of GFP. This makes this line a useful tool for studying B cells as well as T cells. The main advantage of this technique is that now we can study B cells and T cells from LCK GFP zebrafish or we can study B-ALL and T-ALL cells from fish with cancer.These methods enable B cell development and B-ALL studies in zebrafish. Which are relevant to understanding the adaptive immune system and it's malignancies common in pediatric cancer. If you have never done these dissections before you may wish to refine your technique a few times until you can obtain good thymus and marrow samples.Visual demonstration will help viewers to recognize fluorous and B-ALL in fish and to select the correct settings for discriminating B and T cells for their isolation. After anesthetizing with 0.02%Tricaine in fish system water use the GFP filter on an epifluorescence microscope to examine two to six month old zebrafish for fluorescent thymi at the dorsal medial aspect of the branchial cavity. After locating the thymus place euthanized fish in a Petri dish and use the fluorescent microscope to remove the GFP positive thymi.And brightfield microscopy settings to harvest the kidney marrow and spleen. Place each collected organ in a 1.5 milliliter tube containing 500 microliters of cold sorting medium and use a pestle microtube homogenizer to homogenize the tissues on ice. Then, strain the homogenized tissue through a 35 micrometer mesh filter to generate a single cell suspension and place the cells on ice.Beginning at two to four months use a fluorescent microscope to screen double-transgenic rag2:hMYC LCK EGFP fish using low exposure settings to identify bright T cell acute lymphoblastic leukemia or T-ALL and high exposure settings to identify dim B-ALL animals. Wild type control and pre-leukemic fish have GFP localized only in the thymus. Categorize the fish based on the extent of GFP fluorescence using a simple three category system.Level one fish demonstrate a fluorescent signal as a thymic tumor with only a limited local spread. Level two fish demonstrate a fluorescent signal beyond the thymus involving less than 50%of the body. And level three fish demonstrate a fluorescent signal extending beyond 50%of the body.Separate the fish with ALL from those without cancer. And monitor the pre-leukemic fish without GFP positive tumors once monthly for the development of new ALL. For T or B-ALL cell isolation use a razor blade to remove the head and thymic region of a euthanized level three fish and place the body in a petri dish.Use a P-1000 pipette to wash the fish peritoneal cavity with 500 microliters of cold sorting medium, collecting the cells and medium in a five milliliter tube. Using a fresh pipette tip inject two to three additional 200 to 300 microliter volumes of cold sorting medium into the body cavity and use the tip of the pipette to apply gentle pressure to the fish body to extrude the cells out of the body cavity into the five milliliter tube. Then, strain the cell suspension through a 35 microliter mesh filter.And place the cells on ice until their analysis by flow cytometry. For whole body homogenization use a pestle microtube homogenizer to homogenize the body of the fish and add an additional 300 microliters of cold sorting medium to the tube. Filter the cell suspension through a 35 micrometer mesh filter, washing the filter with additional sorting medium as necessary until only tissue debris remains on the filter.Then, place the cells on ice until their analysis. For flow cytometric analysis of the isolated cells set the flow cytometer parameters according to manufacturer's guidelines. And acquire 10, 000 to 50, 000 events to set the forward scatter and side scatter parameters for defining the lymphocyte and progenitor cell gates and to exclude the cellular debris.Exclude the cell doublets and use the lymphoid and/or precursor gates to determine the number and percentage of the GFP positive cells. Using phycoerythrin and the GFP intensities define gates for the GFP negative versus GFP low versus GFP high cells. Learning where to place the gates for isolating the GFP low and GFP high cells is critical to obtaining pure B and T cell populations.Two distinct GFP positive populations, GFP low B cells and GFP high T cells can be obtained from the thymus. B cells residing in the kidney marrow of three month old zebrafish express low levels of GFP. GFP high cells are scarce however, indicating that only a small percentage of T lymphocytes are present in the marrow at three months of age.Likewise, splenic samples show higher percentages of GFP low than GFP high cells. In double-transgenic fish that have not yet developed acute lymphoblastic leukemia, GFP expression in the thymus, marrow and spleen is similar to that observed in single-transgenic LCK EGFP fish. However, the numbers of lymphocytes per organ is increased.Presumably, due to MYC driven expansion of immature B and T cell populations in which the rag2 promoter is active. In double-transgenic fish with B and or T-ALL that have developed fluorescent cancers by six months B-ALL are dimly fluorescent cancers containing mostly GFP low cells. In contrast, brightly fluorescent fish can harbor either isolated T-ALL or mixed populations of both GFP low B-ALL and GFP high T-ALL cells.Indeed, as assessed by quantitative polymerase chain reaction analysis GFP low cells express higher levels of B cell transcripts. And GFP high cells express higher levels of T cell genes, LCK and the GFP marker. It is critical to learn how to recognize fish with B-ALL by microscopy.And how to properly define the flow cytometry gates for separating GFP low and GFP high populations. Following this procedure, you can isolate DNA, RNA, or protein from GFP low and GFP high cells for PCR, sequencing, what's in blood or in vivo transplantation experiments. Once we learned LCK GFB fish have fluorescent B cells in addition to T cells we could study both cell types from the same animal to evaluate B and T-ALL.

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