BIOMEDICAL RESEARCH SUPPORT GRANT

该提案要求支持购买ACAS 570交互式 美国华盛顿大学医学院研究用激光细胞仪 明尼苏达大学。为了进一步实现他们的研究目标， 由目前NIH资助的9名研究人员组成的小组需要仪器 能够：1）荧光定量和分选/细胞选择 原位锚定依赖性细胞群，2）定量 缝隙连接通讯，3）荧光定量 细胞膜光漂白后再分布（FRAP），4） 测定细胞内游离Ca++和pH的变化动力学 在单细胞和贴壁依赖性细胞群中，5）两个和 三维共焦成像只有其中一些需要（4和5）， 大部分是由大学现有的工具来满足的。 明尼苏达Meridian ACAS 570交互式激光细胞仪是唯一一款 商业上可获得的系统，可以进行自动细胞分析， 在未受干扰的组织培养物中分选/选择标记细胞;快速 需要选择性光漂白方法的荧光定量 在间隙连接通讯和FRAP测量分析中， 共聚焦荧光成像与游离Ca++和 细胞内pH测定;以及联合收割机内不同方法 用同样的仪器检测同样的细胞群。拟议文书 将用于细胞内pH、Ca++和缝隙连接分析。 在植入前胚胎中的Ca++通讯;定量 研究所需的粘附于细胞单层的单个血小板 血管损伤;定义结构机制， 血管紧张素II降低肾小球超滤系数 离体肾小球的共聚焦荧光成像;早期定量 与促有丝分裂信号转导相关的游离Ca++增加， 淋巴细胞膜，并确定有丝分裂原诱导的聚集的作用 单核细胞在跨膜信号传导和细胞-细胞 交流在老龄化;分析已知的因素的影响，以增加 酵母毒力对受体表达的细胞为基础;研究 细胞内Ca ~（++）在缝隙连接调节中作用 乳腺癌中的沟通;检查中性粒细胞的能力， 激活内皮细胞单层通过测量Ca++反应， 中性粒细胞产物和测量过氧化物和氢过氧化物， 内皮细胞暴露于中性粒细胞氧化剂;以测量反应性 肾小管上皮细胞中的氧物种，并评估 过氧化氢在细胞间通讯中的作用，细胞内Ca++和 膜流动性;以及从组织培养物中去除不需要的细胞。的 仪器是用户友好的。

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.