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

这个子项目是利用由NIH/NCRR资助的共享仪器赠款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是用于拨款的，而不一定是用于调查人员的机构。描述(由申请人提供)：这项拨款申请请求美国国立卫生研究院支持购买一台BD Biosciences LSR II蓝色激光流式细胞仪，配备四台激光。这一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。我们的申请者小组(四名主要使用者和三名次要使用者)都从事感染免疫学研究，并严重依赖细胞荧光法作为一项主要的实验技术。LSR II是一种高灵敏度和多功能的细胞仪，它将极大地提高我们测量细胞表面和细胞内蛋白表达、体内细胞毒性分析和DNA细胞周期分析的效率。该仪器目前不能在校园内用于BL2/BL3样本。使用这种仪器将有可能从小样本中检测出非常稀有的种群。我们小组的成员研究非常微小的细胞群，如脑浸润性淋巴细胞，非淋巴组织中的记忆CDS T细胞，特殊的巨噬细胞亚群，以及病原体诱导的单个肉芽肿病变。此外，这台仪器将允许我们使用目前的细胞荧光仪无法检测到的新的荧光标记。这些新的标记物很重要，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体内细胞标记和多色感染剂。最后，我们所有人在四色流式细胞术方面都非常有经验；然而，我们的项目将极大地受益于能够从相同的样本中测量更多的参数。目前还不能使用四色细胞仪以相互依赖的方式分析10到14个参数，这对于上面讨论的有限样本大小和实验稀有样本尤其重要，例如从SIV感染的恒河猴制备的淋巴组织或粘膜样本，在这些样本中，我们希望从尽可能少的动物中获得最可能的信息。对感染了包括组织胞浆和锥虫在内的各种病原体的动物的未固定样本的分析将是我们研究计划的真正进步。在过去的几年里，荧光技术的进步取得了爆炸性的进展，特别是在应用于传染病免疫学方面。新一代流式细胞仪分析正在变得可用，我们的用户群想要更进一步。

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.