| Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
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Author:
Date:
2016-12-05
[Abstract] MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are ...
[摘要] MCF10A 3D文化系统提供了腺体乳腺上皮的还原剂模型，其广泛用于研究腺体结构的发育，细胞极性和上皮完整性在上皮细胞功能的控制中的作用以及乳腺癌的机制。在这里我们描述如何使用shRNA筛选方法来识别关键细胞通路，夫妇上皮结构到个别细胞的反应，如细胞周期退出和凋亡。这些研究将有助于询问对早期乳腺肿瘤发生至关重要的遗传变化。该协议描述了设计用于靶向上皮完整性的慢病毒shRNA构建体的文库和用于悬浮MCF10A培养物的慢病毒转导的高效方法。此外，提供的协议设置MCF10A 3D文化在Matrigel的形态和细胞反应研究通过结构化照明和共聚焦显微镜分析免疫染色的三维结构。
关键字： 3D文化，MCF10A，shRNA，上皮完整性，免疫荧光染色，3D成像，形态测量分析
[背景] 上皮细胞形成高度组织的组织结构，其提供物理支持和用于协调细胞信号传导的结构化支架。跨上皮结构的这种协调的信号传导对于上皮生物学是基本的;使得上皮细胞在调节器官大小，形状，功能和基于个体细胞的应答中的动态联合作用（Roignot等人，2013; Shamir和Ewald，2014）。上皮信号传导的联合指挥还提供了一种强有力的肿瘤抑制机制，通过将外部和内部有丝分裂信号门控到静止的上皮组织（Partanen等人，2013; Rejon等人 ...
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| Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues
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Author:
Date:
2016-09-05
[Abstract] Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.
[摘要] 血浆中的铁结合其转运蛋白转铁蛋白，其将铁递送至大多数组织。 在铁过载和某些病理状况下，转铁蛋白的携带能力可能超过，导致非转铁蛋白结合的铁，其优先被肝脏，肾脏，胰腺和心脏摄取。 分别测量组织转铁蛋白和非转铁蛋白结合的铁（分别为TBI和NTBI）在体内的摄取可以通过静脉内施用59 Fe标记的TBI或 NTBI，然后是各种器官的γ计数。 在这里我们描述了测量小鼠组织的TBI和NTBI摄取的详细协议。
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| Chemosensitivity Assay
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Author:
Date:
2013-09-05
[Abstract] Chemoresistance is one of the special properties of cancer stem cells, which is the main cause of chemotherapy failure and plays an important role in the recurrence of various cancers including osteosarcoma. The most widely used assay for evaluating chemoresistance is the modified cell proliferation assay. An equal number of cells are seeded onto 96-well culture plates or 24-well plates, and then different concentrations of anti-cancer drugs are added into each well. After that, measure the cell density or cell activity to observe the effect of anti-cancer drugs to cancer cells or cancer stem cells. The chemoresistance of cells is stronger, the cell density or cell activity is higher. Here, we describe chemosensitivity assays for osteosarcoma cells and osteosarcoma stem-like cells.
[摘要] 化学抗性是癌症干细胞的特殊性质之一，其是化疗失败的主要原因，并且在各种癌症（包括骨肉瘤）的复发中起重要作用。 最广泛使用的用于评价化学抗性的测定法是修饰的细胞增殖测定法。 将等量的细胞接种到96孔培养板或24孔板上，然后将不同浓度的抗癌药物加入每个孔中。 之后，测量细胞密度或细胞活性，观察抗癌药物对癌细胞或癌症干细胞的作用。 细胞的化学抗性更强，细胞密度或细胞活性更高。 在这里，我们描述了骨肉瘤细胞和骨肉瘤干样细胞的化学敏感性测定。
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