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Company: Sigma-Aldrich
Catalog#: H3149
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3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
[Abstract]  Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, ... [摘要]  细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而,在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用,并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性(Edmondson等人,2014)。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型(肿瘤球体)在该领域已经获得了越来越多的普及(Weiswald等人,2015)。使用基质嵌入或封装的球体,悬滴培养的球体,磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中,我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样,该方案也可用于直接从肿瘤组织中分离3D癌细胞系。

【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法(Pampaloni等人,2007; ...

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
[Abstract]  The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal ... [摘要]  从mRNA分子产生蛋白质的效率可以在转录本,细胞类型和细胞状态之间广泛变化。准确测定mRNA翻译效率的方法对获得对转录后基因调控的机理理解至关重要。测量翻译效率的一种方法是确定与mRNA分子相关的核糖体的数目,归一化为编码序列的长度。分析单个mRNA的主要方法是蔗糖梯度分级,其基于结合核糖体的数目物理分离mRNA。在这里,我们描述了精确分析与核糖体的mRNA相关性的简化方案。与以前的方案相比,我们的方法结合内部控制和改进的缓冲条件,共同减少由非特异性mRNA - 核糖体相互作用引起的伪像。此外,我们的直接分数qRT-PCR方案消除了从梯度部分中RNA纯化的需要,这大大减少了所需的手动时间量,并促进了多个条件或基因靶标的并行分析。此外,在该过程中不产生苯酚废物。我们最初开发了协议来研究S-HAC1 mRNA的翻译抑制状态。但是我们还详细介绍了哺乳动物细胞系和组织的适应程序。
【背景】将mRNA翻译成蛋白质是一种高度调节的过程,其可以以不同的速率发生,这取决于基因,细胞环境或环境。翻译起始,延伸和终止的每个步骤可以是最终影响与mRNA相关的核糖体数量的调节点(Dever和Green,2012; ...

Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
[Abstract]  Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO. [摘要]  光感受器是能够响应光以产生视觉信息的专门的视网膜神经元。 在光感受器中,视锥细胞参与颜色辨别和高分辨率中心视觉,并且选择性缺失黄斑变性和视锥营养不良。 这些疾病的可能的治疗溶液是用功能性锥体代替变性细胞。 在这里,我们描述了一个简单的协议,从人类多能干细胞快速生产大量的锥形感光细胞。 分化方案基于使用BMP,TGFβ和WNT拮抗剂COCO的神经诱导的"默认途径"。