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

• Published in: STAR protocols Vol. 3; no. 4; p. 101828
• Main Authors: Qiu, Jiajing, Menon, Vijay, Tzavaras, Nikolaos, Liang, Raymond, Ghaffari, Saghi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2022; Elsevier
• Subjects: Animals; Cell Biology; Cell culture; Cell isolation; Flow Cytometry - methods; Flow Cytometry/Mass Cytometry; Hematopoietic Stem Cells; Humans; Metabolism; Mice; Microscopy; Protocol; Stem Cells; Stem Cells; Cell culture; Microscopy; Flow Cytometry/Mass Cytometry; Metabolism; Cell isolation; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101828

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). [Display omitted] •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.