Bauman J G, de Vries P, Pronk B, Visser J W
Radiobiological Institute, TNO, Rijswijk, The Netherlands.
Acta Histochem Suppl. 1988;36:241-53.
Two procedures to purify and separate pluripotent hemopoietic stem cells (PHSC) and committed progenitor cells from mouse bone marrow cells, and a three colour stem cell staining procedure are described. Visser et al., (J. Exp. Med. 59, 1576-1590, 1984) described the purification of PHSC by metrizamide density gradient centrifugation followed by wheat germ agglutinin-FITC (WGA-FITC) and light scatter sorting using a fluorescence activated cell sorter (FACS). The light density, WGA-positive, high forward (FLS) and low perpendicular light scatter (PLS) blast cells, after removal of the lectin from the sorted cells by the competing sugar, are restained with biotinylated anti-H-2K plus avidin-FITC. The H-2K positive cells proved to be pure pluripotent stem cells. Bauman et al. (J. Cell. Physiol. 128, 133-142, 1986) started by sorting the 6% most positive fluorescent cells with low PLS and high FLS from WGA-FITC stained normal bone marrow. After lectin removal the sorted cells are restained with anti-GM-1.2. Sorted, GM-1.2 negative cells are almost exclusively PHSC plus committed colony forming cells. A three colour staining procedure was designed to measure stem cells in bone marrow samples by flow cytometry. Cells are simultaneously stained with anti-H-2K-biotin plus avidin-phycoerythrin, a rat monoclonal antibody detecting a cell surface antigen plus goat anti rat Ig-FITC and 1-butyryl-pyrene-WGA. Analysis and sorting on the two laser Rijswijk Experimental Light-Activated Cell Sorter (RELACS II) using selected windows indicated that these windows contained high frequencies of PHSC. This multicolour analysis and sorting therefore is equivalent to the multistep sorting procedures.
本文描述了两种从小鼠骨髓细胞中纯化和分离多能造血干细胞（PHSC）及定向祖细胞的方法，以及一种三色干细胞染色方法。维瑟等人（《实验医学杂志》59卷，1576 - 1590页，1984年）描述了通过甲泛葡胺密度梯度离心，随后用麦胚凝集素 - FITC（WGA - FITC）和利用荧光激活细胞分选仪（FACS）进行光散射分选来纯化PHSC。低密度、WGA阳性、高前向（FLS）和低侧向光散射（PLS）的原始细胞，在用竞争糖从分选细胞中去除凝集素后，用生物素化抗H - 2K加抗生物素蛋白 - FITC进行复染。结果证明H - 2K阳性细胞是纯多能干细胞。鲍曼等人（《细胞生理学杂志》128卷，133 - 142页，1986年）首先从WGA - FITC染色的正常骨髓中对6%荧光最强、具有低PLS和高FLS的细胞进行分选。去除凝集素后，对分选细胞用抗GM - 1.2进行复染。分选得到的GM - 1.2阴性细胞几乎全是PHSC加定向集落形成细胞。设计了一种三色染色方法通过流式细胞术检测骨髓样本中的干细胞。细胞同时用抗H - 2K - 生物素加抗生物素蛋白 - 藻红蛋白、一种检测细胞表面抗原的大鼠单克隆抗体加山羊抗大鼠Ig - FITC以及1 - 丁酰芘 - WGA进行染色。在使用选定窗口的两台激光里瑟韦克实验光激活细胞分选仪（RELACS II）上进行分析和分选表明，这些窗口含有高频率的PHSC。因此这种多色分析和分选等同于多步分选程序。
