MBTP 21: Immunophenotyping of hematopoietic stem cells (HSCs) from mouse bone marrow using flow cytometry

Miltenyi Biotec-tested panel 21 (MBTP 21)

This application protocol describes the flow cytometric analysis of hematopoietic stem cells after isolation and enrichment of lineage-negative bone marrow cells from healthy C57BL/6 mice.

Protocol

Gating strategy showing the analysis of HSC subsets from mouse bone marrow. Bone marrow cells were enriched for lineage-negative cells and stained using the previously described panel to analyze HSC subsets. Samples were initially gated on cells using SSC-A/FSC-A gating (A) and on live cells using propidium iodide-negative gating (B). Single cells were gated using FSC-H/FSC-A gating (C). Mouse HSCs are defined by the absence of lineage commitment markers, including CD45R, CD5, CD11b, Ter-119, and Gr-1, and thus are referred to as lineage-negative (Lin) cells (D). Further characterization was done by Sca-1 and CD117 (c-kit) expression (E). Lin Sca-1+ CD117+ cells are also termed LSK cells and include long-term HSCs (LT-HSCs). Two subtypes of mouse LT-HSC can be differentiated either by lack of expression of CD34 and CD135 (F) or by expression of CD150 and lack of expression of CD48 (G), respectively.

Materials

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