Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

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-resolutio...

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Detalles Bibliográficos
Autores principales: Qiu, Jiajing, Menon, Vijay, Tzavaras, Nikolaos, Liang, Raymond, Ghaffari, Saghi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9676629/
https://www.ncbi.nlm.nih.gov/pubmed/36595934
http://dx.doi.org/10.1016/j.xpro.2022.101828
Descripción
Sumario: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 Liang et al. (2020) and Qiu et al. (2021).

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