ABSTRACT
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.
