Cell Cycle Analysis of Hematopoietic Stem Cells

Cell cycle analysis was performed after incubation with Hoechst 33342 and Pyronin Y followed by labeling with FITC-anti-lineage antibodies, APC-anti-c-kit and PE-Cy7-anti-Sca-1 [23 (link)]. BrdU labeling was determined as described [24 (link)]. For Ki67 staining, lineage-depleted cells were first stained with streptavidin-APC-Cy7, APC-anti-c-kit, PE-Cy7-anti-Sca-1, and treated with Cytofix/Cytoperm reagents according to the manufacturer's instructions (BD Biosciences). Cells were incubated with PE-anti-Ki67 antibody and propidium iodide (PI) in BD Perm/Wash buffer and analyzed using FACS.

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Wang W., Yu S., Zimmerman G., Wang Y., Myers J., Yu V.W., Huang D., Huang X., Shim J., Huang Y., Xin W., Qiao P., Yan M., Xin W., Scadden D.T., Stanley P., Lowe J.B., Huang A.Y., Siebel C.W, & Zhou L. (2015). Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem cells (Dayton, Ohio), 33(7), 2280-2293.

Publication 2015

Cytofix/Cytoperm reagents PE-anti-Ki67 antibody BD Perm/Wash buffer

Anti antibodies Anti antibody Brdu Buffer Cell cycle Cells Fitc Hoechst 33342 Perm Propidium iodide Pyronin y Sca 1 Streptavidin

Corresponding Organization : Case Western Reserve University

Other organizations : Harvard University, Massachusetts General Hospital, Harvard Stem Cell Institute, First Affiliated Hospital of Sichuan Medical University, Southwest Medical University, University School, Albert Einstein College of Medicine

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Variable analysis

independent variables

Incubation with Hoechst 33342 and Pyronin Y
Labeling with FITC-anti-lineage antibodies, APC-anti-c-kit and PE-Cy7-anti-Sca-1
BrdU labeling
Staining with streptavidin-APC-Cy7, APC-anti-c-kit, PE-Cy7-anti-Sca-1, and treatment with Cytofix/Cytoperm reagents
Incubation with PE-anti-Ki67 antibody and propidium iodide (PI) in BD Perm/Wash buffer

dependent variables

Cell cycle analysis
BrdU labeling
Ki67 staining

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned.

Annotations

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