Isolation of Primary Mouse Retinal Pigmented Epithelium Cells.
Isolation of Primary Mouse Retinal Pigmented Epithelium Cells.
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DOI：
10.3791/63543
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发表时间：
2022-11-04
期刊：
影响因子：
--
通讯作者：
Tawfik A
中科院分区：
文献类型：
--
作者：
Tomaszewski R;Rajpurohit P;Cheng M;Tawfik A
视网膜色素上皮(RPE)层位于光感受器的正后方，具有复杂的代谢系统，在维持光感受器的功能方面发挥着关键作用。因此，RPE的结构和功能对维持正常视力至关重要。这篇手稿提出了一种建立的小鼠RPE细胞原代分离的方案。RPE分离是研究不同眼病小鼠模型RPE病理基础的分子机制的一个很好的工具。此外，RPE分离有助于比较从野生型和转基因小鼠分离的原代小鼠RPE细胞，以及测试可以加速视力障碍疗法开发的药物。手稿介绍了一种循序渐进的RPE分离方案；从去核到播种的整个过程大约需要4个小时。接种后5-7天内不应更换培养基使分离的细胞不受干扰地生长。这一过程之后，通过免疫荧光对细胞的形态、色素沉着和特定标记进行表征。细胞最多可以传代三到四次。
The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors’ function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn’t be changed for 5–7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.
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