| Experimental Liver Fibrosis and Intrasplenic Transplantation of CD45+ Bone Marrow Cells
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Author:
Date:
2016-10-20
[Abstract] Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. Carbon tetrachloride-induced fibrosis (CCl4) and cirrhosis is one of the oldest, simplest and probably the most widely used toxin-based experimental model for the induction of fibrosis. Here we have explained experimental animal model of liver fibrosis using CCl4, injecting twice a week for a period of 8 weeks. In these fibrotic mice, bone marrow (BM) derived CD45+ cells were transplanted via intrasplenic route ...
[摘要] Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. Carbon tetrachloride-induced fibrosis (CCl4) and cirrhosis is one of the oldest, simplest and probably the most widely used toxin-based experimental model for the induction of fibrosis. Here we have explained experimental animal model of ...
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| Aβ Extraction from Murine Brain Homogenates
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Author:
Date:
2016-04-20
[Abstract] This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals.
[摘要] 该方案详述了从转基因鼠脑匀浆中提取β-淀粉样蛋白（Aβ）。 具体来说，详细说明脑组织的机械均质化和可溶性和不溶性蛋白质的顺序提取。 DEA提取可溶性蛋白质，如Aβ异构体和APP。 甲酸能够提取不溶性蛋白质聚集体，例如与斑块相关的Aβ同种型。 该方法产生可溶性和不溶性提取物，其适于通过western印迹和/或酶联免疫吸附测定（ELISA）分析Aβ物质，并且这些结果有助于评估动物的淀粉样变性负担。
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| Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry
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Author:
Date:
2015-11-05
[Abstract] Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of ...
[摘要] 棕色脂肪组织（BAT）具有显着增加线粒体解偶联燃料氧化以响应肾上腺素刺激的热生成的独特能力。评估棕色脂肪细胞产热能力的关键参数是通过呼吸确定的线粒体解偶联。因此，测量线粒体氧消耗率（OCR）为研究燃料代谢和能量消耗的调节和失调提供了有价值的信息。添加线粒体膜电位的测量允许更加深入地解释呼吸数据。在这里我们提供使用Seahorse XF分析仪测量贴壁完整和质膜透性棕色脂肪细胞呼吸的协议。在方案部分I中，去甲肾上腺素和游离脂肪酸的组合用于诱导解偶联呼吸。然后使用ATP合酶抑制剂寡霉素，化学去偶联剂FCCP和复合物III抑制剂抗霉素A分别测量偶联的，最大的和非线粒体的氧消耗。在方案第II部分中，质膜用重组perfringolysin O透化，胆固醇依赖性细胞溶解素寡聚化成在质膜中专门的孔。这允许代谢物可用性的实验性控制，而不从天然细胞环境中分离线粒体。
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