BD LSR II Blue Laser Flow cytometer

描述（由申请人提供）：此资助申请要求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能够利用荧光技术的最快发展。我们的申请人（四个主要用户和三个次要用户）都从事感染性免疫学研究，并严重依赖细胞荧光测定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，它将大大提高我们测量细胞表面和细胞内蛋白质表达、体内细胞毒性测定和DNA细胞周期分析的效率。该仪器目前在校园内无法用于BL 2/BL 3样品。使用这种仪器将有可能从小样本量中检测非常罕见的人群。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，该仪器将允许我们使用我们目前的细胞荧光计无法检测到的新荧光标记物。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，我们所有人都在四色流式细胞术方面非常有经验;然而，我们的项目将从能够从相同样品中测量更多参数中受益匪浅。以相互依赖的方式分析10至14个参数目前不可能使用4色细胞术，并且对于上述有限的样本量和实验稀有的样本（例如从SIV感染的恒河猴制备的淋巴结或粘膜样本，我们希望从尽可能少的动物中获得尽可能多的信息）都特别重要。对感染了包括组织胞浆菌和锥虫在内的各种病原体的动物的未固定样本进行分析将是我们研究计划的一个真实的进步。在过去的几年里，荧光技术的进步呈爆炸式增长，特别是应用于感染性免疫学。新一代的流式细胞仪检测方法正在变得可用，我们的用户群体希望进入下一个层次。

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.