Isolation and Handling of Mouse Embryonic Fibroblasts
Isolation and Handling of Mouse Embryonic Fibroblasts
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DOI：
10.1007/978-1-4939-1215-5_13
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Liu, Jun
中科院分区：
文献类型：
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作者：
Jain, Kanika;Verma, Paul J.;Liu, Jun
原代小鼠胚胎成纤维细胞（mef）是最常用的饲养层，有助于支持胚胎干细胞（ESC）长期培养的生长和维持多能性。饲料提供维持多能性和防止ESC自发分化所必需的底物/营养物质。由于胚胎成纤维细胞在几次传代后停止分裂，因此必须小心地将它们新鲜地分离出来。在这里，我们提供了一种获得mef的方案，并描述了使用丝裂霉素C治疗使细胞失活的方法。该协议还描述了mef的冷冻、解冻和传代。这个基本方案在我们的实验室运行良好。但是，它可以根据任何用户的特殊要求进行修改和调整。
Primary mouse embryonic fibroblasts (MEFs) are the most commonly used feeder layers that help to support growth and maintain pluripotency of embryonic stem cells (ESC) in long-term culture. Feeders provide substrates/nutrients that are essential to maintain pluripotency and prevent spontaneous differentiation of ESC. Since embryonic fibroblasts stop dividing after a few passages, care must be taken to isolate them freshly. Here, we provide a protocol to derive MEFs and describe the method to inactivate the cells using mitomycin C treatment. The protocol also describes freezing, thawing, and passaging of MEFs. This basic protocol works well in our laboratory. However, it can be modified and adapted according to any user's particular requirement.