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

Jiajing Qiu; Vijay Menon; Nikolaos Tzavaras; Raymond Liang; Saghi Ghaffari

doi:10.1016/j.xpro.2022.101828

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

Jiajing Qiu, Vijay Menon, Nikolaos Tzavaras, Raymond Liang, Saghi Ghaffari

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

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).

Original language	English
Article number	101828
Journal	STAR Protocols
Volume	3
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2022.101828
State	Published - 16 Dec 2022

Keywords

Cell Biology
Cell culture
Cell isolation
Flow Cytometry/Mass Cytometry
Metabolism
Microscopy
Stem Cells

Access to Document

10.1016/j.xpro.2022.101828

Cite this

@article{86310299011b45e099697487a7b3d0c2,

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

abstract = "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{\textquoteright} 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).",

keywords = "Cell Biology, Cell culture, Cell isolation, Flow Cytometry/Mass Cytometry, Metabolism, Microscopy, Stem Cells",

author = "Jiajing Qiu and Vijay Menon and Nikolaos Tzavaras and Raymond Liang and Saghi Ghaffari",

note = "Publisher Copyright: {\textcopyright} 2022 The Authors",

year = "2022",

month = dec,

day = "16",

doi = "10.1016/j.xpro.2022.101828",

language = "English",

volume = "3",

journal = "STAR Protocols",

issn = "2666-1667",

publisher = "Cell Press",

number = "4",

}

TY - JOUR

T1 - Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

AU - Qiu, Jiajing

AU - Menon, Vijay

AU - Tzavaras, Nikolaos

AU - Liang, Raymond

AU - Ghaffari, Saghi

PY - 2022/12/16

Y1 - 2022/12/16

N2 - 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).

AB - 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).

KW - Cell Biology

KW - Cell culture

KW - Cell isolation

KW - Flow Cytometry/Mass Cytometry

KW - Metabolism

KW - Microscopy

KW - Stem Cells

UR - http://www.scopus.com/inward/record.url?scp=85142183823&partnerID=8YFLogxK

U2 - 10.1016/j.xpro.2022.101828

DO - 10.1016/j.xpro.2022.101828

M3 - Article

AN - SCOPUS:85142183823

SN - 2666-1667

VL - 3

JO - STAR Protocols

JF - STAR Protocols

IS - 4

M1 - 101828

ER -

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

Abstract

Keywords

Access to Document

Fingerprint

Cite this