Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing.
Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing.
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DOI：
10.21769/bioprotoc.4243
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发表时间：
2021-12
期刊：
影响因子：
0.8
通讯作者：
Kaustav Mukherjee;J. Bieker
中科院分区：
文献类型：
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作者：
Kaustav Mukherjee;J. Bieker
体内红细胞生成发生在成红细胞岛龛（EBI）中，其包含附着于红系祖细胞并帮助红系祖细胞成熟为成熟网织红细胞的中央巨噬细胞。造血组织如胚胎胎肝中的巨噬细胞是异质的，并表达细胞表面蛋白F4/80。早期从造血组织中分离F4/80+巨噬细胞的方法依赖于FACS分选，但FACS后获得的F4/80+细胞数量相对较低，有时导致RNA质量较差。此外，由于EBI巨噬细胞附着于成红细胞，因此必须小心避免被结合的成红细胞污染。我们已经开发了一种新的方法，用于分离F4/80+细胞从E13.5小鼠胎肝使用磁性纳米粒子，这可以在实验室工作台上进行。在细胞悬浮和均质化过程中，我们还添加了一种肽，该肽可破坏红细胞系巨噬细胞相互作用，并产生无红细胞系污染的F4/80+单细胞。因此，我们的方案产生了富含F4/80+细胞的群体，这些细胞是健康的，并准备好用于敏感技术，如单细胞测序。
In vivo erythropoiesis occurs in the erythroblast island niche (EBI), comprising of a central macrophage that attaches to and aids the maturation of erythroid progenitors into mature reticulocytes. Macrophages in hematopoietic tissue such as embryonic fetal liver are heterogeneous and express the cell surface protein F4/80. Earlier methods of isolating F4/80+ macrophages from hematopoietic tissue relied on FACS sorting, but the relatively low numbers of F4/80+ cells obtained after FACS sometimes led to poor RNA quality. Additionally, since EBI macrophages are attached to erythroblasts, care must be taken to avoid contamination with bound erythroblasts. We have developed a novel method for isolating F4/80+ cells from E13.5 mouse fetal liver using magnetic nanoparticles, which can be performed on the lab bench. During cell suspension and homogenization, we also add a peptide that disrupts erythroid macrophage interactions and generates F4/80+ single cells free of erythroid contamination. Thus, our protocol generates a population enriched in F4/80+ cells that are healthy and ready for sensitive techniques such as single cell sequencing.