Cell Phenotyping Core

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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.