| Isolation of Intestinal Mesenchymal Cells from Adult Mice
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Author:
Date:
2016-09-20
[Abstract] During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells ...
[摘要] 在过去20年间，肠间质细胞（IMC）已经作为重要的细胞类型出现，通过提供结构支持和生长调节元件在肠发育和体内平衡中起着中心作用。 IMC还积极参与伤口愈合反应，从而调节病理状况，例如组织修复，炎症，纤维化和癌发生（Powell等人，2011）。 我们最近已经证明肠间充质特异性信号在肠炎症和癌发生中在体内起重要的生理作用（Koliaraki等人，2012; Roulis等人，/em>。，2014; Koliaraki 。。，2015）。 在这里我们描述了用于从成年小鼠肠道分离和培养间充质细胞的酶法。
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| Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
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Author:
Date:
2016-07-05
[Abstract] mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real-time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in ...
[摘要] mRNA稳定性控制是基因表达的转录后调控中的关键步骤。放线菌素D，一种最初用作抗癌药物的抗生素，由于其对mRNA合成的抑制，已经证明是用于研究细胞中转录物的转换率的方便的工具。在这里，我们描述了添加放线菌素D后的稳定成纤维细胞细胞系，从野生型和三四氯丙胺（TTP）缺陷小鼠胚胎成纤维细胞（MEF）文化，以及一个协议使用半定量实时RT-PCR确定相对转录本丰度。 Northern印迹或NanoString n-Counter是测量mRNA丰度的备选方法，在前一种情况下使用phosphorimager对其进行定量。该方案适合于研究源自转基因小鼠及其各自对照的原代培养细胞和稳定细胞系，并且提供在具有和不具有目标基因的其他相同细胞中的mRNA衰减率的直接比较。
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| Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts
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Author:
Date:
2016-07-05
[Abstract] Fibroblasts are often used as a feeder layer for progenitor or stem cells in co-culture systems. In the heart fibroblasts are important for cardiac development, homeostasis, and remodelling. They provide cardiomyocytes and progenitor cells not only with nutrition but also secrete extracellular matrix that forms the microenvironment that ensures cell survival and function. Although different kinds of mouse fibroblasts have been used in co-cultures (embryonic, skin and cardiac fibroblasts) adult mouse cardiac fibroblasts (AMCFs) create the closest microenvironment to the adult murine heart for culturing adult mouse cardiac progenitor cells. This protocol describes the isolation of cardiac fibroblasts from adult mouse hearts as well as their maintenance in culture.
[摘要] 成纤维细胞通常用作共培养系统中的祖细胞或干细胞的饲养层。 在心脏成纤维细胞对于心脏发育，体内平衡和重塑是重要的。 他们提供心肌细胞和祖细胞不仅与营养，而且分泌细胞外基质，形成微环境，确保细胞的生存和功能。 尽管不同种类的小鼠成纤维细胞已用于共培养（胚胎，皮肤和心脏成纤维细胞），但是成年小鼠心脏成纤维细胞（AMCFs）为培养成年小鼠心脏祖细胞产生了与成年鼠心脏最接近的微环境。 该协议描述了从成年小鼠心脏分离心脏成纤维细胞以及它们在培养物中的维持。
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