This proposal requests support for the purchase of a ACAS 570 Interactive Laser Cytometer for research investigation in the Medical School at the University of Minnesota. To further pursue their research objectives a group of 9 investigators with current NIH funding requires instrumentation capable of: 1) fluorimetric quantitation and sorting/cell selection of anchorage-dependent cell populations in situ, 2) quantitation of gap-junctional communication, 3) quantitation of fluorescence redistribution after photobleaching in cell membranes (FRAP), 4) determination of the kinetics of change in intracellular free Ca++ and pH in single cells and in anchorage-dependent cell populations and, 5) two and three dimensional confocal imaging. Only some of these needs (4 and 5) and mostly partially are met by instruments available at the University of Minnesota. The Meridian ACAS 570 Interactive Laser Cytometer is the only commercially available system that can do automatic cell analysis and sorting/selection of labelled cells in undisturbed tissue culture; rapid fluorimetric quantitation for methods that require selective photobleaching in analysis of gap-junctional communication and FRAP measurements; do confocal fluorescence imaging with ratio analysis for free Ca++ and intracellular pH determinations; and combine different approaches within the same instrument on the same cell populations. The proposed instrument will be used for intracellular pH, Ca++ and analysis of gap-junctional communication in preimplantation embryos; to quantitate changes in Ca++ in individual platelets adherent to cell monolayers needed for investigation of vascular injury; to define the structural mechanisms by which angiotensin II decreases glomerular ultrafiltration coefficient by using confocal fluorescence imaging in isolated glomeruli; to quantitate early increases in free Ca++ associated with mitogenic signal transductions in lymphocyte membranes and to define the role of mitogen-induced aggregation of mononuclear cells in transmembrane signalling and cell-cell communication in aging; to analyze the effects of factors known to augment yeast virulence upon receptor expression on a cell for cell basis; to study the role of intracellular Ca++ in modulation of gap-junctional communication in breast cancer; to examine the ability of neutrophils to activate endothelial cell monolayers by measuring Ca++ responses to neutrophil products and to measure peroxides and hydroperoxides in endothelial cells exposed to neutrophil oxidants; to measure reactive oxygen species in tubular epithelial cells and to assess the effects of hydrogen peroxide in cell-cell communication, intracellular Ca++ and membrane fluidity; and to ablate unwanted cells from tissue culture. The instrument is user-friendly.
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