Core--FACS

分析流式细胞术是参与本项目的所有研究人员的基本技术。这个流式细胞仪核心将通过提供第一个在我们的生物安全3级实验室中安置和操作的流式细胞仪来增强我们机构现有的能力。该机构已经承诺购买该仪器的资金，我们预计在该计划拨款开始之前购买并安装具有低速分选能力的Becton-Dickinson FACSCalibur分析仪。该仪器可对感染强毒或潜在强毒结核分枝杆菌的小鼠细胞悬浮液进行多达四色荧光单细胞分析。使用一系列单克隆抗体进行多色FACS分析，以鉴定和定量特定细胞群，该设备将常规进行，使用装载MHC I类四聚体试剂对特定T细胞群进行染色。通过该核心的建立，还可以在BSL-3环境中分析细胞凋亡、细胞周期进程和钙通量。有限的细胞分选能力应该足以用于拟议的结核分枝杆菌血清学修饰突变体的分离，该核心设施也将成为可能。核心主任Steven Porcelli博士于1989年首次接受分析流式细胞仪的使用培训，并从那时起一直在自己的研究中积极使用这些仪器。他目前也是美国国立卫生研究院资助的AECOM艾滋病研究中心流式细胞术设备的主任，该中心为流式细胞术分析提供了一个BSL-2环境，并使用Becton-Dickinson FACSort仪器进行有限的分选能力。他在培训和监督方面经验丰富

Analytical flow cytometry is an essential technology for all of the investigators participating in this program project. This flow cytometry core will augment existing capabilities at our institution by providing the first flow cytometer to be housed and operated in our Biosafety Level 3 laboratories. Funds for the purchase of this instrument have already been committed by the institution, and we anticipate the purchase and installation of a Becton-Dickinson FACSCalibur analyzer with low speed sorting capability before the initiation of this program grant. This instrument will allow the performance of up to four color fluoresence single cell analysis of cell suspensions harvested from mice infected with virulent or potentially virulent strains of M. tuberculosis. Multicolor FACS analyses using a range of monoclonal antibodies to identify and quantitate specific cell populations will be carried out routinely by this facility, as will staining of specific T cell populations using peptide loaded MHC class I tetramer reagents. Analysis of apoptosis, cell cycle progression and calcium flux will also be available in a BSL-3 environment through the establishment of this core. Limited cell sorting capabilities that should be adequate for the proposed isolation of serologically modified mutants of M. tuberculosis will also by possible with this core facility. The core director, Dr. Steven Porcelli, was first trained in the use of analytical flow cytometers in 1989, and has been actively using these instruments in his own research continuously since that time. He also is currently the director of the flow cytometry facility of the NIH funded AECOM Center for Aids Research, which provides a BSL-2 environment for analytical flow cytometry as well as limited sorting capability using a Becton-Dickinson FACSort instrument. He is experienced in training and supervising technicians and other laboratory personnel to carry out flow cytometry procedures. In establishing this core facility, he will recruit and train an appropriate individual to serve as a dedicated flow cytometry technician to carry out the bulk of the day to day operation of the core.