Awan Aashir, Oliveri Roberto S, Jensen Pernille L, Christensen Søren T, Andersen Claus Yding
Laboratory for Reproductive Biology, University Hospital of Copenhagen, Copenhagen, Denmark.
Methods Mol Biol. 2010;584:195-210. doi: 10.1007/978-1-60761-369-5_11.
This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.
本章介绍了对在无饲养层条件下特定培养的人胚胎干细胞（hESCs）进行免疫荧光（IF）显微镜检查和定量聚合酶链反应（qPCR）分析的步骤。将提供一份详细方案，概述从最初培养细胞、传代至16孔玻璃培养室，到继续进行一般IF和qPCR步骤的各个环节。这些技术将通过转录因子以及刺猬索尼克信号通路、Wnt信号通路和血小板衍生生长因子（PDGF）信号通路的组分在干细胞维持和分化过程中于初级纤毛上的细胞定位的新结果加以说明。此外，还将展示一个qPCR实验样本，用以说明这些技术在解答有关hESC生物学的基本问题方面可成为重要工具。
