Herman Andrew, Greenhough Alexander, Gurevich David B
Faculty of Life Sciences, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK.
Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK.
Methods Mol Biol. 2022;2475:325-337. doi: 10.1007/978-1-0716-2217-9_24.
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.
透明且具有遗传易处理性的斑马鱼越来越被认为是一种有用的模型，可用于对细胞相互作用进行活体成像，并深入了解控制组织稳态、病理和再生的机制。在此，我们描述了一种使用荧光激活细胞分选（FACS）从斑马鱼伤口分离巨噬细胞的方案，以及使用定量实时PCR（qPCR）鉴定表达高水平血管内皮生长因子（vegf）的特定促血管生成巨噬细胞群体的方法。细胞解离和FACS分选技术已针对免疫细胞进行了优化，并成功用于分离伤口内其他荧光标记的群体，如中性粒细胞和内皮细胞。更广泛地说，该方案可以很容易地适用于其他情况，即促血管生成免疫细胞的鉴定对于理解从发育到感染、癌症和糖尿病等病理过程具有变革性意义。
