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Trypsin-EDTA (0.25%), phenol red

胰蛋白酶-EDTA(0.25%),酚红

Company: Thermo Fisher Scientific
Catalog#: 25200056
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3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells
Author:
Date:
2018-04-20
[Abstract]  Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models. We have established an in vitro spheroid formation assay using a 3D co-culture system in which mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ... [摘要]  卵巢癌相当独特,因为卵巢癌细胞可以通过腹膜腔直接分离和扩散。然而,目前还不清楚癌细胞如何在腹膜中存活并形成球状结构。我们最近报道,肿瘤相关巨噬细胞(TAMs)相关的球状体与卵巢癌的临床病理学之间存在很强的相关性,并且TAMs在原位小鼠模型中促进球体形成和肿瘤生长在转移瘤体转移的早期阶段。我们已经建立了使用3D共培养系统的体外球体形成测定法,其中小鼠GFP + F4 / 80 + CD206 F 从含有卵巢癌的供体番茄lysM-cre小鼠的球状体中分离的TAM与在含有2%基质胶的培养基中的ID8细胞(TAM:ID8比例为1:10)混合并接种到用Matrigel预包被的24孔板上。由于transcoelomic转移也与许多其他癌症,如胰腺癌和结肠癌,TAM介导的球体形成实验将提供一个有用的方法来定义卵巢癌和其他transcoelomic转移癌症的分子机制和治疗目标。

【背景】在美国,卵巢癌(OC)是第二常见的妇科癌症和主要死亡原因(Jemal等人,2009; Siegel等人,2012年) )。 OC预后不良的主要原因是腹腔内和盆腔广泛植入转移,通常手术无法完全切除。对腹膜转移现象最广泛的解释是肿瘤细胞在延伸到腹膜表面后与原发肿瘤分离,并在腹膜内播种之前通过腹膜液输送到整个腹腔。有人提出,转移瘤的转移过程可分为几个步骤:1)细胞脱落,失巢凋亡的存活和抵抗; ...

Generation of Luciferase-expressing Tumor Cell Lines
Author:
Date:
2018-04-20
[Abstract]  Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo. For this purpose, the introduction of genes that confer tumors with bioluminescent properties has been a critical advance for oncologic studies in rodents. Methods of introducing bioluminescent genes, such as firefly luciferase, by viral transduction has allowed for the production of tumor cell lines that can be followed in vivo longitudinally over long periods of time. Here we describe methods for the production of stable luciferase expressing tumor cell lines by lentiviral transduction. [摘要]  鼠肿瘤模型对于我们对肿瘤生理学知识和有效肿瘤治疗方法发展的进展至关重要。 这些研究的关键是能够跟踪肿瘤发展并量化体内肿瘤负荷。 为此,引入赋予肿瘤生物发光特性的基因已经成为啮齿动物肿瘤研究的重要进展。 通过病毒转导引入生物发光基因(例如萤火虫萤光素酶)的方法已经允许产生可以在体内纵向长时间地进行的肿瘤细胞系。 在这里我们描述了通过慢病毒转导产生稳定表达荧光素酶的肿瘤细胞系的方法。

【背景】体内跟踪细胞最重要的是能够通过微创方法从外部检测它们。使用来自萤火虫的荧光素酶(Photinus pyralis )的酶促生物发光是用于体内基于图像的细胞追踪的广泛使用的方法。生物发光已被用于各种体内应用,包括报告基因表达的无创成像(Herschman,2004),研究昼夜节律(Southern and Millar,2005),成像脑卒中(Vandeputte

萤火虫荧光素酶氧化物萤光素在分子氧,镁和三磷酸腺苷存在下在560nm产生黄绿色光(Wilson和Hastings,1998; ...

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
Author:
Date:
2018-04-05
[Abstract]  For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones. [摘要]  对于疾病和基础科学研究而言,功能丧失(LOF)突变是非常重要的。 在这里,我们提供了一个简单的流线化协议来产生LOF iPSC系列,通过将CRISPR载体与附加型重编程质粒同时引入成纤维细胞,规避了传统基因编辑和已建立的iPSC系的克隆的技术挑战。 我们的实验已经产生了常染色体基因中所有3种基因型的几乎偶数。 此外,我们提供了一个详细的方法来维护和iPSC克隆的96孔板的基因分型。

【背景】CRISPR / Cas9技术允许简单且特异地针对特定基因组位置进行基因编辑。将该技术与诱导性多能干细胞(iPSC)的疾病建模和再生医学潜力相结合将继续对生物医学研究产生前所未有的影响。然而,使CRISPR / Cas9系统适应iPSC已经提出了几个挑战。在细胞系中进行基因编辑的传统方法是用表达Cas9蛋白质的质粒和指导RNA(gRNA)转染细胞,然后产生单克隆并筛选所需的遗传改变。不幸的是,iPSC不适用于单细胞克隆。已经开发了几种补充媒介和克隆方法来克服这一困难,但仍然充满昂贵的设备(低氧培养箱),困难的技术步骤(FACS分选的单个iPSC的存活)或劳动密集型方案(亚克隆)(Forsyth ,2006; Miyaoka ...

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