Flow Cytometry Core

描述(由申请人提供) 这一至关重要的核心提供了对来自中枢神经系统的脾细胞、颈部淋巴结细胞、外周血淋巴细胞和炎症细胞的常规和一致的分析。正如在个别项目中指出的，对感染期间渗透到中枢神经系统的细胞以及驻留的中枢神经系统细胞(少突胶质细胞、星形胶质细胞和小胶质细胞)的关键分析是整个计划的重要组成部分。通过表面标志物的差异表达来准确确定渗透到中枢神经系统的细胞类型、通过激活状态以及中枢神经系统驻留细胞的身份都是拟议项目的关键。流式细胞术优于免疫组织化学，因为可以通过荧光强度以及多个表面或细胞内标记来区分细胞类型。最好的例子是通过CD45染色的不同强度将小胶质细胞从浸润性血管周围巨噬细胞中分离出来。这一区别不能通过组织学检查来作出。不能通过组织学检查来分析常驻CNS细胞类型。因此，对驻留的CNS细胞类型进行MHC和共刺激分子表达的分析有望具有高度的敏感性。此外，要通过分选获得纯化的种群，需要对其准确身份进行关键评估。所有这些功能都是由这个核心执行的。核心技术人员和核心主任都接受了BD的培训，操作配备了两台激光进行四色分析的FACS Calibur流式细胞仪。

Description (provided by applicant) This critically important core provides routine and consistent analysis of spleen cells, cervical lymph node cells, PBL and inflammatory cells derived from the CNS. As noted in the individual projects, critical analysis of the cells infiltrating the CNS during infection, as well as the resident CNS cells (oligodendroglia, astrocytes and microglia) are important components of the overall program. Differential expression of surface markers for precise determination of the cell types infiltrating the CNS, thru activation state and the identity of CNS resident cells are all critical to the proposed projects. Flow cytometry is superior to immunohistochemistry in that cell types can be distinguished by intensity of fluorescence as well as by multiple surface or intracellular markers. The best example is the separation of microglia from infiltrating perivascular macrophages via their differential intensity of CD45 staining. This distinction cannot be made by histological examination. Analysis of resident CNS cell types cannot be made by histological examination. Analysis of resident CNS cell types for expression of MHC and co-stimulatory molecules thus promises to be highly sensitive. Furthermore, purified populations to be obtained by sorting, require a critical evaluation of their precise identity. All of these functions are carried out by this core. Both the core technician and the core director have been trained by BD in the operation of the FACS Calibur flow cytometer equipped with 2 lasers for 4-color analysis.