REDOX IMAGING INDIVIDUAL EMBRYONIC STEM CELL COLONIES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 氧化还原状态介导胚胎干细胞(ESC)的分化，因此为理解干细胞的多能性提供了重要的补充方法。本项目的目标是对单个胚胎干细胞克隆的线粒体氧化还原状态进行成像，以探讨氧化还原状态与干细胞多能性之间的可能联系，最终开发一种以线粒体氧化还原状态作为细胞分选标记的新型胚胎干细胞分选仪。第一步是在没有任何干扰的情况下对单个小鼠ESC克隆的氧化还原状态进行成像，并将结果与Oct4染色的克隆的免疫化学进行比较。第二步是用另一种胚胎干细胞标记Nanog转染胚胎干细胞，同时成像Nanog的表达和线粒体的氧化还原状态。第三步是用合适的试剂校准氧化还原状态。第四步是使用共聚焦显微镜在单细胞分辨率下对ESC克隆进行成像。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. The goal of this project is to image the mitochondrial redox state of individual embryonic stem cell colonies to investigate the possible association between the redox state and the pluripotency of stem cells to ultimately develop a novel ESC sorter using the mitochondrial redox state as the cell sorting marker. The first step was set to image the redox state of the individual mouse ESC colonies without any disturbance of these colonies and to compare the result with the immunochemistry of the Oct4-stained colonies. The second step was set to transfect the ESC with another embryonic stem cell marker Nanog and simultaneously image the Nanog expression and the mitochondrial redox state. The third step was set to calibrate the redox state with appropriate agents. The fourth step was set to image the ESC colonies at the single cell resolution using a confocal microscope.