Division of Allergy and Clinical Immunology, Jeff and Penny Vinik Center for Allergic Disease Research, Brigham & Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2022;2506:151-165. doi: 10.1007/978-1-0716-2364-0_11.
Brush cells are chemosensory epithelial cells present at most mucosal surfaces.Brush cells are a dominant source of cysteinyl leukotrienes and IL-25 in the airway epithelium and are equipped with the machinery to generate prostaglandins and acetylcholine. Activation of innate type 2 lymphoid cells and dendritic cells triggered by brush cell-derived mediators skew the immune response in the airway to type 2 inflammation that underlies atopic disease such as asthma. This chapter describes an effective method of brush cell isolation from the mouse trachea for transcriptional analysis and from the nasal cavity for transcriptional analysis and ex vivo stimulation.The nasal or tracheal mucosa is first incubated in a dispase solution for easy mechanical separation of the epithelial layer from the underlying submucosa. The detached epithelium is then digested with a papain solution. This method provides high yields of viable brush cells in a single-cell suspension, which can be used for flow cytometric analysis, single-cell sorting, cell culture, and functional assays.In the nose, where brush cells are more abundant, we present two methods of isolation of brush cells: (1) using fluorescent reporter mice that mark brush cells or (2) using a combination of high expression of EpCAM and low expression of CD45 to obtain a population of cells that is enriched for nasal chemosensory brush cells.
刷状细胞是存在于大多数黏膜表面的化学感觉上皮细胞。刷状细胞是气道上皮细胞中半胱氨酰白三烯和 IL-25 的主要来源，并且具有产生前列腺素和乙酰胆碱的机制。刷状细胞衍生的介质激活先天 2 型淋巴样细胞和树突状细胞，使气道中的免疫反应偏向 2 型炎症，这是特应性疾病（如哮喘）的基础。本章描述了一种从小鼠气管中分离用于转录分析的刷状细胞和从鼻腔中分离用于转录分析和体外刺激的刷状细胞的有效方法。首先将鼻或气管黏膜在Dispase 溶液中孵育，以便于将上皮层从下面的黏膜下层机械分离。然后用木瓜蛋白酶溶液消化分离的上皮。该方法可提供大量单细胞悬浮液中的活刷状细胞，可用于流式细胞术分析、单细胞分选、细胞培养和功能测定。在刷状细胞更丰富的鼻腔中，我们提出了两种刷状细胞分离方法：(1) 使用标记刷状细胞的荧光报告小鼠，或 (2) 使用 EpCAM 高表达和 CD45 低表达的组合，以获得富含鼻化学感觉刷状细胞的细胞群体。
