The purpose of the proposed research is to develop methods to simultaneously immunophenotype and molecular phenotype cells to evaluate cytokine receptor and ligand expression. Hematopoietic cells are derived from a small pool of progenitor cells and differentiate from multipotential stem cells into committed progenitor cells that expand to become functional end cells. Antibodies have been produced to some of the proteins on the membrane of hematopoietic cells but not all of them. Since a unique repertoire of proteins is displayed for each subset along the differentiation pathway, investigators seek to be able to separate and identify specific cell populations using flow cytometry. The investigators hypothesize that hematopoietic cell subsets express the unique repertoire of cytokine receptors whose engagement by ligands regulates both proliferation and differentiation of these cells. To understand the functional aspects of this differentiation process it is proposed to develop the procedures necessary to both immunophenotype and molecular phenotype. Molecular phenotyping is a term applied to describe the process of determining specific nucleic acids sequences inside a cell. Both of these methods are necessary because the specific antibodies to identify certain cell types that are available, yet few are available for measuring cytokine ligands and only some ligand receptors. Molecular phenotyping by in situ PCR combined with immunophenotyping is not yet completely reduced to practice, therefore, some development work is also necessary. The detection of nucleic acid sequences will be performed by fluorescence in situ hybridization in suspension as well as by in situ PCR. The proposal emphasizes the use of flow cytometry for detection, however, microscopic imaging will be used as well. The experiments are designed to provide information that will help understand normal hematopoiesis. The development of a reliable method for molecular phenotyping by flow cytometry will have an enormous impact on basic, translational and clinical research. There are three goals to this study. The first is to develop a reliable method for molecular phenotyping by flow cytometry that can be combined with immunophenotyping. The second is to apply this technology to define the specific cytokine receptor expressed by subsets of hematopoietic progenitor cells. The third goal is to define the ligands produced by subsets of T-cells and monocytes. The methods and results will open a new approach to the interrogation of cellular function.
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