| 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 ...
[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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| Measuring the Interactions between Peroxisomes and Chloroplasts by in situ Laser Analysis
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Author:
Date:
2016-04-20
[Abstract] Quantitative analysis has been necessary for deeply understanding characteristic of organelles function. This is the detailed protocol for the quantification of the physical interaction between peroxisomes and chloroplasts taken by laser scanning microscopy described by Oikawa et al. (2015). To clarify the morphological interactions between both organelles, we measured the contact length between two organelles (interaction length) in the fluorescent microscope image by using image analysis software ImageJ. The result clearly revealed that the contact length in light condition is much ...
[摘要] 定量分析对于深入了解细胞器功能的特征是必要的。这是通过由Oikawa等人（2015）描述的激光扫描显微术获取的用于定量过氧化物酶体和叶绿体之间的物理相互作用的详细方案。为了澄清两种细胞器之间的形态学相互作用，我们使用图像分析软件Image J测量荧光显微镜图像中两个细胞器之间的接触长度（相互作用长度）。结果清楚地表明，光照条件下的接触长度比在黑暗的条件。此外，利用飞秒激光和原子力显微镜（AFM）的交叉技术来量化形态相互作用的力。当强激光飞秒激光聚焦在两个细胞器的界面附近时，粘附力由于激光的力而断裂。在光和暗条件下的粘合强度由通过AFM校准的力估计。详细过程在Bio-protocol中描述为另一个名为"Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology"的另一个协议（Hosokawa等人，2016）。这些方法可以应用于不同类型的细胞器如细胞核，线粒体，高尔基体和叶绿体之间的其他物理相互作用。
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| Measurement of the Number of Peroxisomes
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Author:
Date:
2014-11-05
[Abstract] This is the detailed protocol for the measurement of the number of peroxisomes described by Shibata et al. (2013). It is difficult to count the number of organelles in a cell because of the thickness of plant leaves. To overcome this challenge, protoplasts were isolated from leaves, and the number of peroxisomes per protoplast was counted. This method can be applied to other organelles such as mitochondria that are labeled with GFP or its derivatives.
[摘要] 这是用于测量由Shibata等人（2013）描述的过氧化物酶体的数量的详细方案。 由于植物叶片的厚度，很难计数细胞中的细胞器的数目。 为了克服这种挑战，从叶中分离原生质体，并计数每个原生质体的过氧化物酶体的数目。 该方法可应用于用GFP或其衍生物标记的其他细胞器，例如线粒体。
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