Onjiko Rosemary M, Portero Erika P, Moody Sally A, Nemes Peter
Department of Chemistry, George Washington University.
Department of Anatomy & Regenerative Biology, George Washington University.
J Vis Exp. 2017 Dec 22(130):56956. doi: 10.3791/56956.
The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.
对单细胞中的小分子进行定量分析，为更好地理解胚胎发育的基本过程带来了新的可能性。为了能够直接在活胚胎中进行单细胞研究，需要新的分析方法，特别是那些灵敏、有选择性、定量、稳健且能适用于不同细胞大小的方法。在此，我们介绍一种方案，该方案能够对南非爪蟾（非洲爪蟾）自由发育胚胎中的单细胞代谢进行原位分析，非洲爪蟾是细胞和发育生物学中的一种强大模型。这种方法使用毛细管微探针从胚胎中单个已识别的细胞中吸取确定的部分，使相邻细胞保持完整以便后续分析。收集到的细胞内容物通过与高分辨率串联质谱仪联用的微尺度毛细管电泳电喷雾电离（CE-ESI）接口进行分析。这种方法可扩展到各种细胞大小，并与发育中胚胎的复杂三维结构兼容。例如，我们证明微探针单细胞CE-ESI-MS能够阐明在胚胎发育过程中祖细胞产生后代时出现的代谢细胞异质性。除了细胞和发育生物学外，这里描述的单细胞分析方案还适用于其他细胞大小、细胞类型或动物模型。
