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

STAR Protoc. 2022 Dec 16;3(4):101828. doi: 10.1016/j.xpro.2022.101828. Epub 2022 Nov 19.

Authors

Jiajing Qiu¹, Vijay Menon¹, Nikolaos Tzavaras², Raymond Liang³, Saghi Ghaffari⁴

Affiliations

¹ Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
² Department of Neuroscience and Microscopy CoRE, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
³ Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Developmental and Stem Cell Biology Multidisciplinary Training, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁴ Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Developmental and Stem Cell Biology Multidisciplinary Training, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address: saghi.ghaffari@mssm.edu.

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).

Keywords: Cell Biology; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Metabolism; Microscopy; Stem Cells.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Animals
Flow Cytometry / methods
Hematopoietic Stem Cells*
Humans
Mice

Abstract

Publication types

MeSH terms

Grants and funding