Cell Phenotyping Core

该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睾丸的流式细胞术。基于充分开发和验证的技术（超过45个标记物已被验证，包括转录因子和细胞内细胞因子），核心将分析野生型C57 BL/6，Apoe-/-或Ldir-/-小鼠的骨髓细胞（中性粒细胞，单核细胞，巨噬细胞，DC）和淋巴细胞（T细胞亚群，B1 a，B1 b，B2）。我们还将分析动脉周围脂肪组织、主动脉旁淋巴结、远端淋巴结和脾脏中的白细胞含量。具体目标有三：1。为各项目提供白细胞表型分析和白细胞分类; 2.提供内膜和中膜、外膜和主动脉周围脂肪组织中单独的白细胞表型; 3.提供主动脉旁（引流）淋巴结、其他淋巴结和脾脏中的白细胞表型。关键步骤是从小鼠睾丸中获得具有高水平活力（>70%）和几乎完全提取的单细胞悬液。这个程序在每个项目中都要完成，所以在核心中不会使用活的动物。一名经验丰富的技术人员将培训三个项目的博士后和技术人员进行收获。在酶消化步骤期间，将在热控制容器中将所述酶运送至L1 A1。所有的实验室都是本地的，彼此相距不到几分钟。将在LlAl通过FACSCalibur或LSR-II（Becton Dickinson）进行实际流式细胞术。

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.