The function of the Flow Cytometry and Cell Sorting Core (Core D) is to provide each Project with the expertise, equipment, and facilities required for the flow cytometric analyses and cell sorting procedures that are needed for studies of cardiac progenitor cells (CPCs). Accurate identification, characterization, and quantitation of cells in a complex heterogeneous population are technically demanding and require specialized and expensive equipment and personnel. One of the maior advantages of this Program Proiect is that it will make available to all four Proiects the facilities and techniques of flow cytometry and cell sorting. which otherwise would not be affordable to any Proiect in isolation. Flow cytometry-based analysis for the identification of specific cell populations, changes in cell commitment or phenotype, and characterization of cell functions such as cell cycle, viability, signal transduction, or potential, will be crucial to all Projects. A central common facility that performs these technically challenging analyses is therefore indispensable to achieve the goals of the Program Project. Besides the obvious advantages of efficiency and cost savings, the Core will also enable four investigators with diverse and complementary backgrounds (Drs. Rokosh, McCracken, Ratajczak, and O'Toole) to combine their technical and biological expertise in order to ensure that the proposed work is successfully executed. The Core will offer the Program Project investigators the ability to identify and characterize specific cell types from complex populations. Specifically, the Core will perform analysis of marker expression in various cells, including CPCs and more differentiated cells and including changes in marker expression during differentiation;detect intracellular generation of radicals (i.e., using DAFDA for NO and DCFDA for ROS);examine mitochondrial membrane integrity, membrane potential, and function;assess cell viability; determine changes in cell signaling;isolate stem cells utilizing multiple markers;and quantify cells at each phase of the cell cycle. The Core is also active in identifying and characterizing novel cell markers that will be useful for the various Projects.
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