Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing.
Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing.
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DOI：
10.3791/63133
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发表时间：
2021-10-11
期刊：
影响因子：
--
通讯作者：
Hirschi KK
中科院分区：
文献类型：
--
作者：
Chavkin NW;Cain S;Walsh K;Hirschi KK
随着几项研究揭示了血管生物学中的新范式，新一代测序技术的最新进展促进了研究人员对分子和细胞生物学的了解。将这些方法应用于血管发育模型需要优化从胚胎和出生后组织中分离细胞的技术。细胞产量、活力和纯度都需要达到最大，才能从下一代测序方法中获得准确和可重复性的结果。研究人员使用新生小鼠视网膜血管模型来研究血管发育的机制。研究人员已经使用这个模型来研究血管形成和成熟过程中血管生成和动静脉命运指定的机制。应用下一代测序技术研究视网膜血管发育模型，需要优化视网膜内皮细胞的分离方法，使细胞产量、活性和纯度最大化。本协议描述了一种使用荧光激活细胞分选(FACS)分离、消化和纯化小鼠视网膜组织的方法。结果表明，经流式细胞仪纯化的CD31+/CD45−内皮细胞群内皮细胞基因表达高度丰富，60min内皮细胞存活率无明显变化。包括使用该方法分离的内皮细胞的下一代测序方法的代表性结果，包括批量RNA测序和单细胞RNA测序，表明这种用于视网膜内皮细胞分离的方法与下一代测序应用相兼容。这种视网膜内皮细胞分离方法将使先进的测序技术能够揭示血管发育的新机制。该方案描述了一种分离小鼠出生后视网膜内皮细胞的方法，该方法针对细胞产量、纯度和活性进行了优化。这些细胞适用于下一代测序方法。
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. This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for Next-Generation Sequencing approaches.
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DOI：
10.1007/978-1-4939-9240-9_2
发表时间：
2019-01-01
期刊：
SINGLE CELL METHODS: SEQUENCING AND PROTEOMICS
影响因子：
--
作者：
Braga, Felipe A. Vieira;Miragaia, Ricardo J.
通讯作者：
Miragaia, Ricardo J.
影响因子：
16.6
作者：
Fang JS;Coon BG;Gillis N;Chen Z;Qiu J;Chittenden TW;Burt JM;Schwartz MA;Hirschi KK
通讯作者：
Hirschi KK
影响因子：
--
作者：
Green, Michael R;Sambrook, Joseph
通讯作者：
Sambrook, Joseph
影响因子：
64.5
作者：
Benedito, Rui;Roca, Cristina;Adams, Ralf H.
通讯作者：
Adams, Ralf H.
影响因子：
3.6
作者：
dela Paz, Nathaniel G.;D'Amore, Patricia A.
通讯作者：
D'Amore, Patricia A.