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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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
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author Qiu, Jiajing
Menon, Vijay
Tzavaras, Nikolaos
Liang, Raymond
Ghaffari, Saghi
author_facet Qiu, Jiajing
Menon, Vijay
Tzavaras, Nikolaos
Liang, Raymond
Ghaffari, Saghi
author_sort Qiu, Jiajing
collection PubMed
description 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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spelling pubmed-96766292022-11-22 Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging Qiu, Jiajing Menon, Vijay Tzavaras, Nikolaos Liang, Raymond Ghaffari, Saghi STAR Protoc Protocol 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). Elsevier 2022-11-19 /pmc/articles/PMC9676629/ /pubmed/36595934 http://dx.doi.org/10.1016/j.xpro.2022.101828 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Qiu, Jiajing
Menon, Vijay
Tzavaras, Nikolaos
Liang, Raymond
Ghaffari, Saghi
Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title_full Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title_fullStr Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title_full_unstemmed Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title_short Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
title_sort protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging
topic Protocol
url 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
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