Core--FACS

分析流式细胞术是参与该计划项目的所有研究人员的基本技术。该流式细胞仪核心将通过提供第一台流式细胞仪来增强我们机构的现有能力，该流式细胞仪将在我们的生物安全3级实验室中安装和运行。该机构已经承诺购买该仪器的资金，我们预计在启动该计划拨款之前购买和安装具有低速分选能力的Becton-Dickinson FACSCalibur分析仪。该仪器将允许对从感染M的毒性或潜在毒性菌株的小鼠收获的细胞悬液进行多达四色荧光单细胞分析。结核该机构将常规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，并使用负载肽的MHC I类四聚体试剂对特定T细胞群进行染色。通过建立该核心，也可在BSL-3环境中分析细胞凋亡、细胞周期进程和钙流。有限的细胞分选能力，应足以用于分离血清学修饰的M突变体。结核病也将有可能通过这一核心设施。核心主任Steven Porcelli博士于1989年首次接受了使用分析流式细胞仪的培训，自那时以来，他一直在自己的研究中积极使用这些仪器。他目前也是NIH资助的AECOM艾滋病研究中心流式细胞术设施的主任，该中心为分析流式细胞术提供了BSL-2环境，并使用Becton-Dickinson FACSort仪器提供了有限的分选能力。他在培训和监督方面经验丰富 技术人员和其他实验室人员进行流式细胞术程序。在建立该核心设施的过程中，他将招募并培训一名合适的人员，作为专门的流式细胞术技术员，负责核心的大部分日常操作。

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.