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

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.