Funds are requested for purchase of a Coherent Dye Laser System and a Coulter Extended Data Analysis System (EASY 88) for upgrading an Epics V Fluorescent Activated Cell Sorter (FACS) currently in operation in a multiuser facility at The University of Virginia. The Dye Laser System will provide dual laser capabilities thereby permitting the use of multiple antibody labeling techniques using Texas red-fluorescein (phycoerythrin) dye combinations, as well as permitting many multicolor analysis and sorting protocols which are not possible with the single laser systems presently available due to overlap of emission spectra. The EASY 88 System involves the addition of an Intel 8088 micro- processor-based computer system, expanded memory, a separate graphics processor and color monitor, and a separate monochrome monitor for program interaction. This system is necessary given the added complexity of data analysis with the dual-laser system, and will improve our data analysis capabilities by permitting user programing (at present we are limited to using software provided by Coulter). The requested upgrade components will support ongoing, federally-funded research of 7 investigators in 5 Departments at The University of Virginia. Ongoing operational support for the Facility is provided by an Institutional award by user-generated- fees. The research proposals described herein include: (1) studies of growth control and differentiation in vascular smooth muscle using dual laser cytometric analysis of contractile proteins, protein-DNA content, and DNA-antibody staining; (2) dual antibody analysis of myosin heavy chain expression during skeletal muscle development; (3) selection of cell surface defective mutants of Chlamydomonas utilizing multicolor FACS; (4) multicolor immunoanalysis of surface antigens in studies of mechanisms of T-lymphocyte antigen recognition; (5) multicolor immunolabeling and combined DNA-antibody labeling for sorting X- and Y-chromosome containing sperm for controlling gender and for sorting decidual cells; (6) use of multicolor immunolabeling to characterize aberrant lymphocyte and mononuclear cells in diabetes; and (7) use of multicolor immunolabeling to characterize pre-B cell inducing activity in leukemia research. Investigators have an immediate need for the requested instrumentation for a major component of their research programs. The requested upgrade will enable investigators to pursue approaches currently closed to them as well as improving techniques compromised on our present system.
