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

MBTP 21: Miltenyi Biotec-tested panel 21

This application protocol describes the flow cytometric hematopoietic stem cell identification and analysis after isolation and enrichment of lineage-negative bone marrow cells from healthy C57BL/6 mice using the Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin (# 130-092-613).

Protocol

Antibody panels for hematopoietic stem cells

Antibody panel 1

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin-Cell type exclusion--
BiotinREA746-VioBlue®450/50 (violet)
CD34REA383Cell type exclusionFITC525/50 (blue)
CD135 (FLT3)REA779Cell type exclusionAPC655-730 (red)
Sca-1REA422LSK cellsPE-Vio® 770750LP (blue)
CD1173C11*LSK cellsPE585/40 (blue)
Propidium Iodide Solution-Viability-655-730 (blue)

*Alternative REAfinity™ Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.

Antibody panel 2

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin-Cell type exclusion--
BiotinREA746-VioBlue450/50 (violet)
CD48HM48-1*Cell type exclusionFITC525/50 (blue)
CD150 (SLAM)REA299Long-term HSCAPC655-730 (red)
Sca-1REA422LSK cellsPE-Vio 770750LP (blue)
CD1173C11*LSK cellsPE585/40 (blue)
Propidium Iodide Solution-Viability-655-730 (blue)

*Alternative REAfinity Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.

Gating strategy

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.

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Materials

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