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Ham's F-12 Nutrient Mix

Company: Thermo Fisher Scientific
Catalog#: 11765054
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Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
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Date:
2017-09-05
[Abstract]  Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further ... [摘要]  大多数上皮肿瘤已经显示含有可能是肿瘤进展和转移的驱动力的癌症干细胞(Kreso和Dick,2014; Nassar和Blanpain,2016)。 为了深入研究这些细胞,依赖于细胞表面标志物或荧光报告基因的细胞分离策略是必不可少的,分离策略必须保持其活力。 从大部分肿瘤中分离不同细胞群的能力将继续加深我们对癌症干细胞生物学的认识。 在这里,我们报告了结合机械肿瘤解离,酶处理和流式细胞术的策略,从其天然微环境中分离出纯种群的上皮癌干细胞。 该技术可用于进一步功能性地分析癌症干细胞(RNA测序和表观遗传学分析),在培养物中培养它们或在移植测定中直接使用它们。
【背景】肿瘤复发和转移是大多数与癌症有关的死亡的主要原因。恶性肿瘤可能由干细胞群体启动和维持(Nassar和Blanpain,2016; Bonnet和Dick,1997),这些细胞是预防复发的重要治疗靶点(Baumann et al。,2008)。研究表明,鳞状细胞癌由肿瘤干细胞亚群维持,其抗药性,并通过进行自我更新和分化(如正常干细胞)引发肿瘤复发,产生增殖祖细胞,其分化形成肿瘤大部分(Locke et al。,2005; Prince et al。,2007; Malanchi et al。,2008; de Sousa e Melo et ...

Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells
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Date:
2017-09-05
[Abstract]  Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED increases resolution compared to confocal microscopy, which can be further improved by image processing using deconvolution software. [摘要]  人肺上皮A549细胞中的呼吸道合胞病毒(RSV)感染诱导丝状伪足,由F-肌动蛋白组成的细胞突起,延伸至相邻的未感染细胞(Mehedi等,2016)。 通过受激发射耗尽(STED)显微镜的高分辨率成像显示沿着这些丝状伪足的丝状RSV颗粒,表明丝状伪足有助于RSV细胞对细胞的扩散(Mehedi等,2016)。 在本协议中,我们描述如何修复,渗透,免疫染色和挂载RSV感染的A549细胞进行STED成像。 我们显示与共聚焦显微镜相比,STED增加了分辨率,可以通过使用去卷积软件的图像处理进一步改进。
【背景】RSV形成多形性病毒颗粒,其长度大约为直径约100nm,长度大约为10μm(Bachi和Howe,1973; Mehedi等,2016)。高分辨率光学显微技术是可视化RSV感染细胞和病毒颗粒之间相互作用的关键。在最近的一项研究中,我们使用超分辨率荧光显微镜来研究人肺上皮A549细胞中的RSV细胞对细胞的扩散。
  STED显微镜是超分辨率显微镜技术之一,已被开发以规避约200nm衍射屏障的光限制(Hell和Wichmann,1994; Westphal等人,2008)。 ...

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
Author:
Date:
2017-06-05
[Abstract]  Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an ... [摘要]  基于CRISPR的可编程RNA引导核酸酶(集群定期交织的短回文重复)-Cas(CRISPR相关蛋白)系统已被应用于各种类型的细胞作为强大的基因组编辑工具。通过使用激活诱导的胞苷脱氨酶(AID)代替CRISPR / Cas9系统的核酸酶活性,我们开发了一种用于靶向核苷酸替代(C至T或G至A)的基因组编辑工具,无供体DNA模板(图1 ; Nishida等人,2016)。这里我们描述Target-AID在哺乳动物细胞基因组中进行可编程点突变的详细方法。在这里描述了用于靶向中国仓鼠卵巢(CHO)细胞中的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶(HPRT)基因的具体方法作为实例,而该方法主要应适用于任何感兴趣的基因广泛的细胞类型。


图1. Target-AID及其可靶向位点的示意图。在指导RNA(gRNA)依赖性方式中,通过接头与nCas9(D10A)融合的PmCDA1在-21周围进行可编程胞苷突变至相对于哺乳动物细胞中非互补链上的PAM序列的-16位。可目标地点是根据以前的工作中观察到的有效的基础替代(> 20%)来确定的。
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