Fluorescence-Activated Cell Sorting and Quantitative Real-Time PCR to Reveal VEGF-Expressing Macrophage Populations in the Zebrafish Larvae

Andrew Herman, Alexander Greenhough, David B Gurevich

Research output: Chapter or section in a book/report/conference proceedingChapter or section

Abstract

The transparent, genetically tractable zebrafish is increasingly recognized as a useful model to both live image and uncover mechanistic insight into cell interactions governing tissue homeostasis, pathology, and regeneration. Here, we describe a protocol for the isolation of macrophages from zebrafish wounds using fluorescence-activated cell sorting (FACS), and the identification of specific pro-angiogenic macrophage populations that express high levels of vascular endothelial growth factor (vegf) using quantitative real-time PCR (qPCR). The cell dissociation and FACS sorting techniques have been optimized for immune cells and successfully used to isolate other fluorescently marked populations within the wound such as neutrophils and endothelial cells. More broadly, this protocol can be easily adapted to other contexts where identification of pro-angiogenic immune cells is transformative for understanding, from development to pathologies such as infection, cancer, and diabetes.

Original languageEnglish
Title of host publicationVEGF Signalling
Subtitle of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages325-337
Number of pages13
Volume2475
ISBN (Electronic)9781071622162
ISBN (Print)9781071622179
DOIs
Publication statusE-pub ahead of print - 21 Apr 2022

Publication series

NameMethods in Molecular Biology
Volume2475
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • FACS
  • Immune cells
  • Macrophages
  • Quantitative PCR
  • VEGF
  • Wounding
  • Zebrafish

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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