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Recombinant human epidermal growth factor

hEGF

Company: Sigma-Aldrich
Catalog#: E9644
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Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts
Author:
Date:
2020-11-20
[Abstract]  

With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary

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[摘要]  [摘要]随着CRISPR-Cas的出现以及能够轻松修饰各种生物的基因组的能力,越来越多地开发大鼠模型来询问乳腺发育和肿瘤发生的遗传事件。已经为小鼠和人类组织彻底开发了用于分离和表征乳腺上皮细胞亚群的方案,但是对大鼠特异性方案的需求却在不断增长。迄今为止,还没有用于分离大鼠乳腺上皮亚群的标准方案。分析大鼠乳腺层次的变化将有助于我们阐明乳腺癌中的分子事件,乳腺癌亚型的起源细胞以及肿瘤微环境的影响。在这里,我们描述为1)大鼠乳腺上皮细胞分离开发的几种方法;2)大鼠乳腺成纤维细胞分离;3)培养大鼠乳腺上皮细胞;通过4)流式细胞仪分析和鉴定大鼠乳腺细胞;5)免疫荧光。源自该协议的细胞可用于多种目的,包括RNAseq ,药物研究,功能测定,基因/蛋白质表达分析和图像分析。


[背景]大多数与乳腺有关的研究都是在小鼠模型和人体样品中进行的。然而,由于其具有类似于人的药代动力学特征和乳腺发育,该疾病的大鼠模型正变得越来越流行(Russo等人,1990;Jiunn等人,2008; Smalley等人,2016)。像人类腺癌一样,大鼠乳腺癌也经历组织学发展阶段(Russo等,1990; Singh等,2000),并且是卵巢激素依赖性的(Thompson等,1998; ...

Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes
Author:
Date:
2018-01-20
[Abstract]  Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture of multiple types of stem/progenitor cells, we recently established a novel method to generate bipotent liver progenitor cells, named chemically induced liver progenitors (CLiPs), from adult rat MHs. Here, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat MHs and then describe how to induce CLiPs from these MHs. In addition, we describe a method to evaluate the bipotentiality of ... [摘要]  原代成熟肝细胞(MH)或其祖细胞是重症肝病中细胞移植治疗的候选细胞来源。然而,这些细胞的稳定培养或多能干细胞的等效细胞的产生受到限制。我们使用先前在多种类型的干/祖细胞稳定培养中发现有用的小分子混合物,最近建立了一种从成年大鼠MHs产生双能肝脏祖细胞(命名为化学诱导肝祖细胞(CLiPs))的新方法。在这里,我们描述了诱导大鼠CLiPs的详细方案。我们首先描述分离原代鼠MH的方法,然后描述如何从这些MH中诱导CLiPs。另外,我们描述了一种评估产生的CLiPs分化成肝细胞和胆管上皮细胞的双能性的方法。我们还介绍了如何通过长期的文化和详细的示例数据建立稳定的CLiP。可以在2周内产生初级CLiPs,并且可以在2.5-4个月内建立经历10次传代的稳定的CLiPs,批次间变异性。
【背景】对于实现肝病再生医学的新型细胞来源有着强烈的需求。目前唯一的治疗终末期肝病的方法是肝移植,但是由于供者短缺,其应用受到限制。最近,我们小组提出了一种产生能够在体外稳定地扩增的新型LPC的方法,并且可以以广泛的效率重新繁殖慢性肝炎动物模型的损伤肝脏(Katsuda等人, / ...

Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
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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