神经血管单元(NVU)，由内皮细胞、包绕在微血管周围的平滑肌细胞、毗邻星形胶质细胞和微胶质细胞与神经元组成，对中枢神经系统的正常功能至关重要。NVU对血脑屏障(BBB)功能进行关键调节，而这种功能在多种神经系统疾病中受损，因此成为关键的治疗靶点。为了了解BBB功能障碍的程度和细胞来源，需要同时隔离和分析NVU细胞。本文介绍了一种基于流式细胞术的EPAM-ia方法的操作步骤，用于同时隔离和分析内皮细胞、平滑肌细胞、星形胶质细胞和微胶质细胞。该方法基于酶、机械均质和针对每种细胞类型特定的过滤，然后结合免疫染色和荧光激活细胞分选技术来进行不同NVU细胞类型的差异处理。细胞分类策略包括细胞特异性和排除标记，以隔离主要的NVU细胞类型。该方案需要6小时左右处理一对或两对动物。隔离的NVU细胞可用于以转录组学、蛋白质组学和细胞培养为基础的下游应用。多种细胞类型分析可应用于与BBB功能障碍相关的神经系统疾病中，以从单个或多个NVU细胞类型中获得稳健的靶点。EPAM-ia方法还适用于隔离其他几种细胞类型，包括癌细胞和免疫细胞。该方案适用于小鼠和人类来源的健康和病理组织，并可同其他类似方案进行比较。 ©2023 Springer Nature Limited。

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.© 2023. Springer Nature Limited.