The use of flow cytometry for the purification of normal hematopoietic progenitor and stem cells as well as to analyze the expression and function of defined cell surface and intracellular molecules expressed by hematopoietic cells as central to the projects in this grant. Flow cytometry can be used to analyze and purify viable and fixed cells based on the expression of cell surface and intracellular determinants, physical properties (cell size determined by light scattering), and labeling with vital dyes. Flow cytometry also can be used to study the simultaneous expression of cell surface determinants and intracellular molecules including proteins and mRNA, as well as DNA content for cell cycle analysis. The Cell Separation Core will perform all cell analysis and cell sorting, for both bulk and single cell deposition (cloning), using flow cytometry. Flow cytometry will be used by each of the projects in this grant to purify progenitor and stem cells for direct in vitro and in vivo studies and to study expression of cell surface and intracellular molecules by stem cells and their progeny. Therefore, the function of the Cell Separation Core facility will be to perform multiple parameter flow cytometric analysis of cell lines and cultured and infected primary cells and multi-parameter cell sorting and single cell cloning to isolate subsets of human, mouse and baboon marrow and blood cells as well as stem and progenitor cells necessary for in vitro.
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