{"id":107399,"date":"2022-04-21T15:33:33","date_gmt":"2022-04-21T19:33:33","guid":{"rendered":"https:\/\/med.virginia.edu\/cell-biology\/?p=107399"},"modified":"2026-05-21T13:00:18","modified_gmt":"2026-05-21T17:00:18","slug":"isolation-of-murine-retinal-endothelial-cells-for-next-generation-sequencing","status":"publish","type":"post","link":"https:\/\/med.virginia.edu\/cell-biology\/2022\/04\/21\/isolation-of-murine-retinal-endothelial-cells-for-next-generation-sequencing\/","title":{"rendered":"Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing"},"content":{"rendered":"

UVA Author:<\/strong> Nicholas Chavkin
\nCitation:<\/strong> Chavkin NW, Cain S, Walsh K, Hirschi KK. Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing. J Vis Exp. 2021;(176):10.3791\/63133. Published 2021 Oct 11. doi:10.3791\/63133<\/p>\n

DOI:<\/strong> https:\/\/doi.org\/10.3791\/63133<\/a>
\nPub-Med Number<\/strong>: 34694293<\/p>\n

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Recent improvements in next-generation sequencing have advanced researchers’ knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+\/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.<\/p>\n<\/div>\n<\/div>\n

<\/div>\n","protected":false},"excerpt":{"rendered":"

UVA Author: Nicholas Chavkin Citation: Chavkin NW, Cain S, Walsh K, Hirschi KK. Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing. J Vis Exp. 2021;(176):10.3791\/63133. Published 2021 Oct 11. doi:10.3791\/63133 DOI: https:\/\/doi.org\/10.3791\/63133 Pub-Med Number: 34694293 Recent improvements in next-generation sequencing have advanced researchers’ knowledge of molecular and cellular biology, with several studies revealing novel […]<\/p>\n","protected":false},"author":1740,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":"","_links_to":"","_links_to_target":""},"categories":[13],"tags":[21],"class_list":["post-107399","post","type-post","status-publish","format-standard","hentry","category-featured-publications","tag-delete"],"acf":false,"yoast_head":"\nIsolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing - Department of Cell Biology<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/med.virginia.edu\/cell-biology\/2022\/04\/21\/isolation-of-murine-retinal-endothelial-cells-for-next-generation-sequencing\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing - Department of Cell Biology\" \/>\n<meta property=\"og:description\" content=\"UVA Author: Nicholas Chavkin Citation: Chavkin NW, Cain S, Walsh K, Hirschi KK. 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