BD LSR II BLUE LASER FLOW CYTOMETER: T CELLS IN GRANULOMATOUS IMMUNE RESPONSES

该子项目是利用由NIH/NCRR资助的共享仪器补助金提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是资助机构，不一定是研究者的机构。描述（由申请人提供）：本拨款申请要求NIH支持购买带有四个激光器的BD Biosciences LSR II蓝色激光流式细胞仪。这种非常复杂的设备将使我们的用户群体能够利用荧光技术的最快发展。我们的申请者组（4个主要用户和3个次要用户）都从事感染免疫学研究，并且严重依赖细胞荧光法作为主要的实验技术。LSR II是一种高灵敏度和多功能的细胞仪，它将极大地提高效率，我们可以测量细胞表面和细胞内蛋白质的表达，体内细胞毒性测定，并进行DNA细胞周期分析。该仪器目前在校园内不能用于BL2/BL3样品。使用这种仪器可以从小样本量中发现非常罕见的种群。我们小组的成员研究非常微小的细胞群，如脑浸润淋巴细胞、非淋巴组织中的记忆CDS T细胞、特化巨噬细胞亚群和病原体诱导的单个肉芽肿病变。此外，该仪器将使我们能够使用我们目前的细胞荧光仪无法检测到的新的荧光标记物。这些新的标记物是重要的，因为它们将允许使用新的多色转基因或逆转录病毒介导的细胞和多色感染因子的体内标记。最后，我们对四色流式细胞术都很有经验；然而，如果能够从相同的样本中测量更多的参数，我们的项目将受益匪浅。目前使用四色细胞术无法以相互依赖的方式分析10到14个参数，这对于上述有限的样本量和实验罕见的样本（例如从SIV感染的恒河猴制备的淋巴结或粘膜样本）尤其重要，因为我们希望从尽可能少的动物中获得尽可能多的信息。分析来自感染了多种病原体（包括组织浆体和锥虫）的动物的未固定样本将是我们研究计划的真正进步。荧光技术在过去几年中取得了突飞猛进的发展，特别是在感染免疫学方面的应用。新一代的流式细胞术检测正在变得可用，我们的用户组希望推进到下一个水平。

This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): This grant application requests NIH support for the purchase of a BD Biosciences LSR II Blue Laser flow cytometer with four lasers. This very sophisticated piece of equipment will allow our user group to take advantage of the fastest developments in fluorescent technology. Our group of applicants (four major users and three minor users) all work on infectious immunology and rely heavily on cytofluorimetry as a major experimental technology. The LSR II is a highly sensitive and versatile cytometer which will vastly improve the efficiency with which we can measure expression of cell surface and intracellular proteins, assay for in vivo cytotoxicity, and perform DNA cell cycle analysis. This instrument is not currently available on campus for use with BL2/BL3 samples. Use of this instrument will make it possible to detect very rare populations from small sample sizes. Members of our group study very minute cell populations such as brain-infiltrating lymphocytes, memory CDS T cells in nonlymphoid tissue, specialized macrophage subsets, and pathogen-induced single granuloma lesions. In addition, this instrument would allow us to use new fluorescence markers that cannot be detected by our current cytofluorimeters. These new markers are important because they will allow the use of new multicolor transgenic or retrovirus-mediated in vivo labeling of cells and multi-color infectious agents. Finally, all of us are very experienced in 4-color flow cytometry; however, our projects would benefit greatly from being able to measure more parameters from the same samples. The analysis of 10 to 14 parameters in an interdependent way is not currently possible using 4-color cytometry, and is particularly important for both the limiting sample sizes discussed above and samples of experimental rarity, such as lymph nodes or mucosal samples prepared from SIV infected rhesus macaques where we want the most possible information from the least possible number of animals. Analysis of unfixed samples from animals infected with diverse pathogens including Histoplasma and Trypanosoma would be a real advance to our research programs. Advances in fluorescence technology have exploded in the past few years, especially as applied to infectious immunology. A new generation of flow cytometric assays is becoming available and our group of users would like to advance to the next level.