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Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish
Published on: February 22, 2019
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Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish
Jessica Burroughs-Garcia1, Ameera Hasan1, Gilseung Park1
1Section of Pediatric Hematology-Oncology, Department of Pediatrics, University of Oklahoma Health Sciences Center.
Journal of Visualized Experiments : Jove
|March 12, 2019
Summary
Researchers developed a new protocol to purify zebrafish B cells using the lck:eGFP transgenic model. This method enables detailed studies of B cell development and related malignancies in zebrafish.
Area of Science:
- Immunology
- Developmental Biology
- Zebrafish Models
Background:
- Zebrafish (Danio rerio) possess adaptive immunity with B and T lymphocytes, making them valuable for studying lymphopoiesis.
- Studying zebrafish B cell development is challenging due to a lack of specific cell surface markers and antibodies.
- Existing transgenic models like lck:eGFP are primarily used for T cell studies.
Purpose of the Study:
- To develop a reliable method for purifying B-lineage cells from zebrafish.
- To enable comprehensive research into zebrafish B cell development and associated diseases.
- To overcome limitations in current zebrafish immunology research.
Main Methods:
- Utilized the lck:eGFP transgenic zebrafish line, discovering that B cells express GFP at lower levels than T cells.
- Developed a protocol employing fluorescent-activated cell sorting (FACS) for B cell isolation.
- Applied the method to various tissues including marrow, thymus, spleen, and blood.
Main Results:
- Successfully purified B-lineage cells from lck:eGFP zebrafish, including double-transgenic lines.
- Demonstrated that low-level GFP expression in B cells allows their differentiation from high-GFP expressing T cells.
- Established a protocol applicable to multiple zebrafish tissues.
Conclusions:
- The developed FACS protocol provides a novel approach for isolating zebrafish B cells.
- This method significantly advances the study of B cell development and B-lymphocyte malignancies in zebrafish.
- The lck:eGFP model can now be effectively used for B cell research, expanding its utility.
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