Ualiyeva Saltanat, Boyd Amelia A, Barrett Nora A, Bankova Lora G
Division of Allergy and Clinical Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Bio Protoc. 2021 Sep 20;11(18):e4163. doi: 10.21769/BioProtoc.4163.
Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and assays. Graphic abstract: Workflow of nasal digestion for brush cell isolation.
孤立化学感受上皮细胞散布于大多数黏膜表面。在肠道黏膜中它们被称为簇状细胞，在气管中被称为刷状细胞，在鼻黏膜中被称为孤立化学感受细胞和微绒毛细胞。它们是上皮中白细胞介素-25的主要来源，也参与乙酰胆碱的生成。我们最近证明，鼻孤立化学感受（刷状）细胞在受到钙离子载体、气传变应原以及危险相关分子（如ATP和UTP）刺激时，能够产生大量的半胱氨酰白三烯，并且这一机制依赖于嘌呤能受体P2Y2在刷状细胞上的表达。本方案描述了一种在小鼠中分离鼻刷状细胞的有效方法。该方法基于鼻腔黏膜层的物理分离以及用分散酶预孵育，随后用木瓜蛋白酶溶液消化。通过这种方式获得的单细胞悬液含有高产量的刷状细胞，可用于荧光激活细胞分选（FACS）、RNA测序及检测。图形摘要：用于刷状细胞分离的鼻腔消化流程。
