Adipose Tissue Core - Columbia

脂肪组织核心将提供各种各样的具有成本效益的服务， 人和小鼠脂肪组织。服务将包括细胞分离和分选技术， 组织学、分子生物学和蛋白质化学。特别是，新鲜分离的人和小鼠 将脂肪样品进行胶原酶消化（在最不可能改变基因的条件下 表达）以将原代脂肪细胞与基质血管部分分离。脂肪组织 随后将巨噬细胞与基质血管部分分离 磁珠将在整个脂肪组织及其细胞中分析基因表达。 使用定量“实时”逆转录酶聚合酶链反应（rt-PCR， Roche LightCycler）。RNA拷贝数的定量将来自质粒标准品 每个感兴趣基因的曲线。基因表达分析将通过以下分析来补充： 蛋白质表达的关键脂肪细胞分泌产物的蛋白质印迹。此外，西方 印迹法将用于研究胰岛素信号通路的各种组分的活化 在各种体内条件下取样的脂肪组织中。定量评估 巨噬细胞向脂肪组织中的浸润将通过两种替代方法进行： 定量rt-PCR（使用特异性巨噬细胞标志物）和荧光激活细胞分选 （FACS）分析。由于许多上述技术需要新鲜获得的脂肪组织， 将这些技术集中在一个核心中将有利于效率并使处理最小化。 以下是核心提供的几种额外的高度专业化的技术。脂肪细胞 在脂肪组织样品的锇固定后，将对大小和数量进行定量。串行 大鼠脂肪组织的活组织检查将由Vasselli博士在体内进行。脂蛋白脂酶活性将 由约翰逊博士在冷冻脂肪组织样本中进行测定。重组脂肪因子生产 将由劳伦斯·夏皮罗医生提供

The Adipose Tissue Core will provide a wide variety of cost-effective services for the study of human and mouse adipose tissue. Services will include techniques of cell separation and sorting, histology, molecular biology and protein chemistry. Specifically, freshly isolated human and mouse fat samples will be subjected to collagenase digestion (under conditions least likely to alter gene expression) to separate primary adipocytes from the stromal vascular fraction. Adipose tissue macrophages will subsequently be separated from the stromal vascular fraction with antibodycoated magnetic beads. Gene expression will be analyzed in whole adipose tissue and its cellular components using quantitative 'real-time' reverse-transcriptase polymerase chain reaction (rt-PCR, Roche LightCycler). Quantification of RNA copy number will be derived from plasmid standard curves for every gene of interest. Analysis of gene expression will be complemented by analysis of protein expression of key adipocyte secretory products by Western blotting. Additionally, Western blotting will be used to study the activation of various components of the insulin signaling pathway in adipose tissue sampled under a variety of in vivo conditions. Quantitative assessment of macrophage infiltration into adipose tissue will be performed by two alternative methods: quantitative rt-PCR (using specific macrophage markers) and Fluorescence Activated Cell Sorting (FACS) analysis. Since many of the above techniques require freshly obtained adipose tissue, the concentration of these techniques in one Core should favor efficiency and minimize handling. The following are several additional, highly specialized techniques offered by the Core. Adipocyte size and number will be quantified following osmium fixation of adipose tissue samples. Serial biopsies of rat adipose tissue will be performed in vivo by Dr. Vasselli. Lipoprotein lipase activity will be assayed in frozen adipose tissue samples by Dr. Johnson. Recombinant adipokine production will be provided by Dr. Lawrence Shapiro.