从鼠喉中收集单个上皮细胞的方法。
Method for Collecting Single Epithelial Cells from the Mouse Larynx.
机构信息
Department of Otolaryngology - Head & Neck Surgery, School of Medicine, Stanford University, Stanford, California, U.S.A.
Department of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, U.S.A.
出版信息
Laryngoscope. 2024 Feb;134(2):786-794. doi: 10.1002/lary.30970. Epub 2023 Aug 21.
OBJECTIVE
The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells.
METHODS
Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, β-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types.
RESULTS
We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters.
CONCLUSION
We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury.
LEVEL OF EVIDENCE
NA Laryngoscope, 134:786-794, 2024.
目的
喉由特化的上皮细胞群体构成。研究单个上皮细胞类型中发生的分子变化需要一种可靠的方法从喉中分离这些细胞。我们的目标是开发一种从小鼠喉中分离单个上皮细胞的方法，同时最大限度地减少来自非喉部位和非上皮性喉细胞的污染。
方法
处死小鼠，小心暴露并分离喉与非喉部位。将一个小牙刷插入喉入口并旋转以获取上皮细胞。将细胞转移到收集培养基中，计数，并对喉上皮（即泛角蛋白、EpCAM、NGFR、p63、K5、β-微管蛋白、MUC5AC）和非上皮（即波形蛋白）细胞标志物进行细胞旋涂制备染色。对刷取的喉组织切片进行组织病理学检查，以评估细胞采集的深度。进行初步单细胞 RNA 测序（scRNA-seq）以确认该方法可以捕获多种喉细胞类型。
结果
我们从单个喉中收集了 6000-8000 个细胞，从三个组织的刷取物中组合收集了 35000-40000 个细胞。组织病理学显示刷取去除了喉的上皮层和一些下层组织。免疫荧光染色显示收获细胞的表型主要为上皮细胞。成功进行了初步的 scRNA-seq，显示了九个独特的细胞簇。
结论
我们开发了一种从小鼠喉中分离单个上皮细胞的可靠方法。该方法将有助于收集各种下游细胞和分子分析的喉细胞，包括 scRNA-seq、蛋白质分析和基于细胞培养的实验，用于喉损伤后。
证据水平
无喉镜，134：786-794，2024。
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