Del Río-Jay Yalén, Barthelaix Audrey, Reyes-Martínez Cristian, Duperray Christophe, Solis-Cascante Camila J, Hidalgo Yessia, Luz-Crawford Patricia, Djouad Farida, Feijoo Carmen G
Fish Immunology Laboratory, Facultad Ciencias de la Vida, Universidad Andrés Bello, Avenida República 330, Santiago 8370186, Chile.
Institute for Regenerative Medicine & Biotherapy (IRMB), Centre Hospitalier Universitaire de Montpellier Hôpital Saint Eloi, University Montpellier, INSERM, 80 Avenue Augustin Fliche, 34295 Montpellier, France.
Methods Protoc. 2024 May 17;7(3):43. doi: 10.3390/mps7030043.
Intestinal macrophages have been poorly studied in fish, mainly due to the lack of specific molecular markers for their identification and isolation. To address this gap, using the zebrafish (:EGFP) transgenic line, we developed a fluorescence-activated cell sorting strategy (FACS) that allows us to isolate different intestinal macrophage subpopulations, based on GFP expression and morphological differences. Also, we achieved the purification of high-quality total RNA from each population to perform transcriptomic analysis. The complete strategy comprises three steps, including intestine dissection and tissue dissociation, the isolation of each intestinal macrophage population via FACS, and the extraction of total RNA. To be able to characterize molecularly different macrophage subpopulations and link them to their functional properties will allow us to unravel intestinal macrophage biology.
鱼类肠道巨噬细胞的研究较少，主要原因是缺乏用于其鉴定和分离的特异性分子标记。为了填补这一空白，我们利用斑马鱼（:EGFP）转基因品系，开发了一种荧光激活细胞分选策略（FACS），该策略使我们能够根据绿色荧光蛋白（GFP）的表达和形态差异分离不同的肠道巨噬细胞亚群。此外，我们从每个亚群中获得了高质量的总RNA，用于进行转录组分析。完整的策略包括三个步骤，即肠道解剖和组织解离、通过FACS分离每个肠道巨噬细胞亚群以及总RNA的提取。能够从分子水平上表征不同的巨噬细胞亚群并将它们与其功能特性联系起来，将有助于我们揭示肠道巨噬细胞生物学。
