Cai Xiongwei, Zheng Yi, Speck Nancy A
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.
Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center.
J Vis Exp. 2018 Aug 22(138):56855. doi: 10.3791/56855.
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.
造血干细胞（HSCs）是稀有细胞，小鼠骨髓中仅含有约25,000个具有表型的长期重建造血干细胞。优化了一种蛋白质免疫印迹方法，该方法适用于分析少量造血干细胞（500 - 15,000个细胞）。对具有表型的造血干细胞进行纯化、精确计数，并直接在Laemmli样品缓冲液中裂解。含有等量细胞的裂解物通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析，然后按照标准蛋白质免疫印迹方法制备和处理印迹。使用该方法，通常可以分析2,000 - 5,000个造血干细胞，在某些情况下，从低至500个细胞中也能获得数据，而大多数出版物报道的细胞数量为20,000至40,000个。该方法应普遍适用于其他造血细胞，并能够使用标准实验室程序对少量细胞进行常规分析。
