| Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells
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Author:
Date:
2016-05-20
[Abstract] Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and solve this kind of issue. Moreover PLA experiments are read out using fluorescence or bright field microscopy, providing additional information on intracellular interactions localization significantly bettering classical IP procedures.
PLA reagents are made of complementary small oligonucleotides “minus” and “plus” probes which specifically recognize host species from the primary antibodies (Abs) targeting the two proteins you are ...
[摘要] 蛋白质 - 蛋白质相互作用实验，例如共免疫沉淀（IP）测定，经典地需要大量的细胞。当您的工作关注稀有细胞群体（例如淋巴细胞亚型）时，这成为一个问题。 O-link Bioscience开发了邻近连接测定（PLA）试剂和程序以缓解和解决这类问题。此外，使用荧光或明视场显微镜读出PLA实验，提供关于细胞内相互作用定位的额外信息，显着改善经典IP程序。 PLA试剂由互补的小寡核苷酸"负"和"正"探针组成，其特异性识别来自靶向您感兴趣的两种蛋白质的一抗（Abs）的宿主物种。实验必须使用来自不同物种，小鼠或山羊）作为PLA探针"减"或"加"对一抗的特定宿主物种（例如，"加"抗兔与"减"抗鼠或"加" "如果使用小鼠和兔初级Abs，则允许使用"减"抗兔组合的抗小鼠）。当两种PLA探针足够接近（40nm）时，在连接酶孵育后发生连接，产生环DNA。接下来，由于在该步骤中掺入的聚合酶和互补荧光核苷酸，这些形成环的DNA被扩增。每个发光点此后被认为是两种蛋白质之间的相互作用位点（图1）。
图1. Duolink ®实验的示意图。 ...
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| Colon Cancer-associated Fibroblast Establishment and Culture Growth
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Author:
Date:
2016-04-05
[Abstract] Cancer-associated fibroblasts (CAFs) are one of the major players in tumor-stroma crosstalk. Findings in experimental studies suggest important roles for CAFs in regulation of tumor growth, metastasis and drug response (Hanahan and Coussens, 2012). Furthermore, their clinical relevance is supported by new findings from tumor analyses, demonstrating the prognostic and response-predictive significance of CAF-derived markers or gene signatures (Berdiel-hacer et al., 2014; Finak et al., 2008; Navab et al., 2011; Paulsson and Micke, 2014). CAFs are a heterogeneous pool of cell subsets with distinct functions which needs to be better defined by their marker expressions. The development of a methodology for the establishment of fibroblast primary cultures derived from ...
[摘要] 癌症相关成纤维细胞（CAF）是肿瘤 - 基质串扰的主要参与者之一。实验研究的结果表明CAFs在肿瘤生长，转移和药物反应的调节中的重要作用（Hanahan和Coussens，2012）。此外，它们的临床相关性得到来自肿瘤分析的新发现的支持，表明CAF衍生的标记或基因特征的预后和应答预测显着性（Berdiel-hacer等人，2014; Finak 等人，2008; Navab等人，2011; Paulsson和Micke，2014）。 CAF是具有不同功能的细胞亚群的异质池，其需要由它们的标记表达更好地定义。开发用于建立源自人结肠肿瘤的成纤维细胞原代培养物的方法允许我们表征其功能和分子性质（Herrera等人，2013）。此外，CAF影响肿瘤生长和转移的不同分子机制仍有待澄清。因此，癌症相关成纤维细胞的功能和分子表征是完全了解他们在肿瘤进展中的作用的必要条件。
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| siRNA Screening for Genes Involved in HSV-1 Replication
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Author:
Date:
2014-08-20
[Abstract] Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, ...
[摘要] 小干扰RNA（siRNA）是能够沉默转录后基因表达的小（通常为18-24个核苷酸）RNA分子，因此，它们提供了一种简单的方法，通过其可以容易地评估单个基因在复杂细胞系统中的作用。 由于siRNA易于实验使用，并且可以设计为靶向任何基因（包括病原体），它们的使用完全适合于并且容易地适应于高通量全基因组筛选方法学和一系列表型测定。 这里我们描述了使用大的siRNA文库（> 8,000个基因单独靶向）来筛选和鉴定功能上涉及人疱疹病毒（1型单纯疱疹病毒; HSV-1）（Griffiths）的复制的宿主因子， et al。，2013; Griffiths，2013）。
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