Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging.
Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging.
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DOI：
10.1016/j.xpro.2022.101828
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发表时间：
2022-12-16
期刊：
影响因子：
--
通讯作者：
Ghaffari, Saghi
中科院分区：
文献类型：
--
作者：
Qiu, Jiajing;Menon, Vijay;Tzavaras, Nikolaos;Liang, Raymond;Ghaffari, Saghi
线粒体膜电位（MMP）分离高度纯化的造血干细胞（HSC）内功能不同的亚群。在这里，我们详细介绍了一个协议，流式细胞仪分离MMP亚组分的表型定义的小鼠和人类HSC。这些步骤之后是HSC溶酶体的高/超分辨率免疫荧光显微镜检查。虽然该方案描述了静止HSC的分离，这是最有效的子集，但它也可以应用于其他HSC子集。该方案克服了与低HSC数相关的一些实验挑战。有关本方案使用和执行的完整详细信息，请参见和。使用线粒体膜电位分离具有不同效力的HSC亚群使用同上μ-载玻片对罕见HSC亚群进行免疫荧光染色使用共聚焦/超分辨率显微镜对HSC细胞器进行成像基于共聚焦图像分析HSC中的溶酶体含量和通量活性出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。线粒体膜电位（MMP）分离高度纯化的造血干细胞（HSC）内功能不同的亚群。在这里，我们详细介绍了一个协议，流式细胞仪分离MMP亚组分的表型定义的小鼠和人类HSC。这些步骤之后是HSC溶酶体的高/超分辨率免疫荧光显微镜检查。虽然该方案描述了静止HSC的分离，这是最有效的子集，但它也可以应用于其他HSC子集。该方案克服了与低HSC数相关的一些实验挑战。
Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs’ lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol, please refer to and. Using mitochondrial membrane potential to isolate HSC subsets with distinct potency Immunofluorescence staining of rare HSC subpopulations using ibid μ-slides Imaging HSC organelles using confocal/super-resolution microscopy Analyzing lysosomal content and flux activity in HSCs based on confocal images Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs’ lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers.
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影响因子：
4.8
作者：
Schieke, Stefan M.;Ma, Mingchao;Finkel, Toren
通讯作者：
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DOI：
10.1073/pnas.1915905116
发表时间：
2019-11-05
影响因子：
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DOI：
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发表时间：
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期刊：
Science (New York, N.Y.)
影响因子：
--
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通讯作者：
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影响因子：
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作者：
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