Czupalla Cathrin J, Yousef Hanadie, Wyss-Coray Tony, Butcher Eugene C
Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
Bio Protoc. 2018 Nov 20;8(22). doi: 10.21769/BioProtoc.3092.
The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. In the past, approaches to isolate endothelial cells have either involved the use of transgenic reporter mice or suffered from insufficiently pure cell populations and poor yield. This protocol here is based on well-established protocols that were modified and combined to allow single cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after careful removal of the meninges and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested in two steps resulting in a single cell suspension. Cells are stained with a cocktail of fluorochrome-conjugated antibodies identifying not only brain endothelial cells, but also potentially contaminating cell types such as pericytes, astrocytes, and lineage cells. Using flow cytometry, cell populations are separated and sorted directly into either RNA lysis buffer for bulk RNA analyses (., RNA microarray and RNA-Seq) or in pure fetal bovine serum to preserve viability for other downstream applications such as single cell RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq). The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 3 × 10 cells from a pool of four adult mice.
脑内皮是一种高度特化的血管结构，维持着中枢神经系统（CNS）的活性和完整性。先前的研究报道，在众多神经病理学中脑内皮的完整性都会受到损害。因此，建立一种方法使研究人员能够结合疾病小鼠模型研究和了解中枢神经系统内皮细胞的分子变化及潜在机制，就显得尤为重要。过去，分离内皮细胞的方法要么涉及使用转基因报告小鼠，要么存在细胞群体纯度不足和产量低的问题。这里的方案是基于已确立的方案进行修改和组合而成的，通过荧光激活细胞分选（FACS）实现高度纯净的脑内皮细胞群体的单细胞分离。简要地说，在小心去除脑膜并解剖皮质/海马后，将脑组织进行机械匀浆并分两步进行酶消化，得到单细胞悬液。细胞用荧光染料偶联抗体混合物染色，这些抗体不仅能识别脑内皮细胞，还能识别潜在的污染细胞类型，如周细胞、星形胶质细胞和谱系细胞。使用流式细胞术，将细胞群体分离并直接分选到用于大量RNA分析（如RNA微阵列和RNA测序）的RNA裂解缓冲液中，或分选到纯胎牛血清中以保持活力，用于其他下游应用，如单细胞RNA测序和转座酶可及染色质测序分析（ATAC-Seq）。该方案不需要转基因表达来标记脑内皮细胞，因此可应用于任何小鼠模型。在我们的操作中，该方案具有高度可重复性，从四只成年小鼠的脑组织中平均可获得3×10个细胞。
