We are requesting funds to purchase the """"""""Anchored Cell Analysis and Sorting"""""""" (ACAS) system marketed as the ACAS 470 by Meridian Instruments, Okemos, Michigan. This instrument performs the functions of the fluorescence-activated cell sorter (FACS) but on adherent cells. Although FACS, based on flow cytofluorimetry, is a valuable tool for studying cell differentiation and function of non-adherent cells, it suffers from serious deficiencies when applied to adherent cells, the subjects of study for many investigators in Wistar. When adherent cells are prepared as single-cell suspensions for FACS many molecules are stripped from their surfaces, and cell-cell and cell-substratum interactions are disrupted. This limits the range of molecules available as cell markers and has consequences that are difficult to determine for the subsequent behavior of sorted cells. The ACAS 470 avoids these problems by using a microscope equipped with a motor-driven stage, laser optics and a fluorescence detection system, controlled by a micro-computer, to scan and analyze a field of cells stained in situ with fluorescent antibodies or other fluorescent probes (e.g., for DNA). Particular living cells can be repeatedly observed over extended periods so that their individual behaviors can be recorded. The laser beam can be used to kill cells automatically according to pre-set criteria, providing a sorting capacity equivalent to the FACS. The ACAS can also be used in a photobleaching method to follow the redistribution of molecules within cells, or between cells, providing an automated assay for gap junction communication. We propose to use the ACAS for a variety of investigations, including the study of cell differentiation, changes in growth factor receptors in aging fibroblasts, the behavior of endothelial cell lines, and the processing of viral antigens, and for the isolation of cell lines from spontaneous and experimentally induced tumors.
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