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.
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
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping