PUBLICATION
Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish
- Authors
- Burroughs-Garcia, J., Hasan, A., Park, G., Borga, C., Frazer, J.K.
- ID
- ZDB-PUB-190312-11
- Date
- 2019
- Source
- Journal of visualized experiments : JoVE (144): (Journal)
- Registered Authors
- Frazer, Kimble
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- B-Lymphocytes/cytology*
- B-Lymphocytes/pathology*
- Cell Differentiation
- Cell Lineage
- Cell Separation/methods*
- Green Fluorescent Proteins/genetics*
- Humans
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology
- T-Lymphocytes/cytology
- Zebrafish/genetics
- Zebrafish/immunology*
- PubMed
- 30855581 Full text @ J. Vis. Exp.
Citation
Burroughs-Garcia, J., Hasan, A., Park, G., Borga, C., Frazer, J.K. (2019) Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish. Journal of visualized experiments : JoVE. (144):.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping