The following monoclonal antibodies were used to perform staining of Lin–/Sca-1+/c-Kit+ (SKL) cells and Lin–/Sca-1+/CD45+ (HSCs): FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend) and PE-Cy5-anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage markers (Lin) were purchased from BD Biosciences: anti-CD45R/B220 (clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T-cell receptor β (clone H57-597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3) and anti-CD45 (clone 30-F11) and conjugated with PE as described.24 (link), 29 (link), 31 (link), 32 (link) All monoclonal antibodies were added at saturating concentrations, and the cells were then incubated for 30 min on ice, washed twice, resuspended in RPMI-1640 plus 2% fetal bovine serum, and analyzed with an LSR II flow cytometer (BD Biosciences, San Diego, CA, USA).
Protocol detail
Immunophenotyping of Hematopoietic Stem Cells
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FITC–anti-CD117
PE-Cy5-anti-mouse Ly-6 A/E
anti-Gr-1 (clone RB6-8C5),
anti-TCRγδ (clone GL3)
anti-CD45 (clone 30-F11)
LSR II flow cytometer
Cd11b
Cells
Clone
Fetal bovine serum
Fitc
Hscs
Monoclonal antibodies
Mouse
Sca 1
T cell receptor β
Tcrγδ
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Variable analysis
independent variables
- Monoclonal antibodies used for staining
- Flow cytometer used for analysis
- Staining pattern of Lin-/Sca-1+/c-Kit+ (SKL) cells
- Staining pattern of Lin-/Sca-1+/CD45+ (HSCs)
- Incubation time (30 min on ice)
- Washing steps (twice)
- Cell resuspension in RPMI-1640 plus 2% fetal bovine serum
- No positive controls were explicitly mentioned.
- No negative controls were explicitly mentioned.
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