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J Vis Exp. 2018 Aug 22;(138). doi: 10.3791/56855.

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells.

Author information

1
Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center; Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania; Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University.
2
Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center.
3
Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania; nancyas@upenn.edu.

Abstract

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

PMID:
30199018
PMCID:
PMC6231698
[Available on 2019-08-22]
DOI:
10.3791/56855
[Indexed for MEDLINE]

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