Cell Phenotyping Core

这一核心的目的是为所有三个项目提供主动脉流式细胞术和荧光激活细胞分选服务。PI的实验室开发了用于小鼠主动脉的流式细胞术。基于完全开发和验证的技术(包括转录因子和细胞内细胞因子在内的45种标志物已得到验证)，核心将分析野生型C57BL/6、APOE-/-或Ldir-/-小鼠主动脉壁中的髓细胞(中性粒细胞、单核细胞、巨噬细胞、DC)和淋巴细胞(T细胞亚群，B1A、B1B、B2)。我们还将分析动脉周围脂肪组织、腹主动脉旁淋巴结、远端淋巴结和脾中的白细胞含量。有三个具体目标：1.为所有项目提供小鼠主动脉的白细胞表型和分选；2.在内膜和中层、外膜和主动脉周围脂肪组织中提供单独的白细胞表型；3.在腹主动脉旁(引流)淋巴结、其他淋巴结和脾中提供白细胞表型。关键的步骤是从小鼠的主动脉中获得单细胞悬液，具有高水平的活性(&gt；70%)和几乎完全的提取。这一程序在每个项目中都会完成，因此核心不会使用活动物。一名经验丰富的技术人员将培训这三个项目的博士后和技术人员。在酶消化步骤中，主动脉将在一个受热控制的容器中被运送到L1Al。所有实验室都是本地的，而且彼此之间只有几分钟的距离。FACSCalibur或LSR-II(Becton Dickinson)的实际流式细胞术将在LA1进行。

The purpose of this core is to provide aortic flow cytometry and fluorescence-activated cell sorting services to all three projects. The Pi's lab developed flow cytometry for mouse aortas. Based on fully developed and validated technology (more than 45 markers have been validated, including transcription factors and intracellular cytokines), the core will analyze myeloid cells (neutrophils, monocytes, macrophages, DCs), and lymphocytes (T cell subsets, B1a, B1b, B2) in the wall of aortas of wild-type C57BL/6, Apoe-/- or Ldir-/- mice. We will also analyze the leukocyte content in periarterial adipose tissue, in paraaortic lymph nodes, in distant lymph nodes and in the spleen. There are three specific aims: 1. To provide leukocyte phenotyping and sorting of mouse aortas to all projects; 2. To provide separate leukocyte phenotyping in intima and media, adventitia, and periaortic adipose tissue; 3. To provide leukocyte phenotyping in paraaortic (draining) lymph nodes, other lymph nodes and spleen. The key procedure is obtaining a single cell suspension from mouse aortas, with a high level of viability (>70%) and nearly complete extraction. This procedure is done in each ofthe projects, so no live animals will be used in the core. An experienced technician will train postdocs and technicians from the three projects in harvesting. During the enzymatic digestion step, the aortas will be transported to LlAl in a thermally controlled container. All labs are local and within a few minutes of each other. The actual flow cytometry by FACSCalibur or LSR-II (Becton Dickinson) will be done at LlAl.