一种用于分离涡虫干细胞和神经细胞的消减荧光激活细胞分选法。

A Subtractive FACS Method for Isolation of Planarian Stem Cells and Neural Cells.

作者信息

Hayashi Tetsutaro, Agata Kiyokazu

机构信息

Genome Resource and Analysis Unit, Center for Developmental Biology, RIKEN, Kobe, Hyogo, Japan.

Bioinformatics Research Unit, Advanced Center for Computing and Communication, RIKEN, Wako, Saitama, Japan.

出版信息

Methods Mol Biol. 2018;1774:467-478. doi: 10.1007/978-1-4939-7802-1_19.

DOI:10.1007/978-1-4939-7802-1_19

PMID:29916172

Abstract

Planarians are among the metazoan organisms with the greatest regenerative abilities. This ability is based on their pluripotent stem cells, called neoblasts, which constitute 10-20% of the cells in their body. Elucidating the molecular mechanisms of the planarian stem cell system, for example, the maintenance of stem cell homeostasis and orchestration of lineage choices, contributes powerfully to the advancement of regenerative biology. Our group has developed fluorescence activated cell sorting (FACS) methodologies for the reliable isolation of planarian stem cells, which constitutes an important experimental asset in the field. Here, we describe detailed protocols for the isolation of (1) planarian stem cells and (2) neural cells. Planarian stem cells are isolated by subtraction of the FACS profiles of intact and γ-ray-irradiated (= stem cell depleted) animals stained with Hoechst 33342 and Calcein AM. The neural cells are isolated by subtracting the FACS profiles of head and tail fragments stained with Hoechst 33258 and Merocyanine 540.

摘要

涡虫是后生动物中具有最强再生能力的生物之一。这种能力基于它们的多能干细胞，即新细胞，其占涡虫体内细胞的10%-20%。阐明涡虫干细胞系统的分子机制，例如干细胞稳态的维持和谱系选择的调控，对再生生物学的发展有极大的推动作用。我们团队已经开发出荧光激活细胞分选（FACS）方法，用于可靠地分离涡虫干细胞，这是该领域一项重要的实验资源。在此，我们描述了分离（1）涡虫干细胞和（2）神经细胞的详细方案。通过减去用Hoechst 33342和钙黄绿素AM染色的完整涡虫和γ射线照射（=干细胞耗尽）涡虫的FACS图谱来分离涡虫干细胞。通过减去用Hoechst 33258和部花青540染色的头部和尾部片段的FACS图谱来分离神经细胞。