| Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants
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Author:
Date:
2017-08-05
[Abstract] The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.
[摘要] 秀丽隐杆线虫种系是胚芽干细胞研究的重要模型系统。 野生型C。 线虫生殖细胞是合胞体，因此不能在体外培养中分离。 相比之下，来自肿瘤突变体的生殖细胞可以完全被细胞化并从突变动物中完整分离。 在这里，我们描述了从肿瘤突变体中分离生殖细胞的详细方案，其允许生殖细胞在体外原代培养中长时间维持。 该协议已从2016年Chaudhari等人改编。
【背景】秀丽生殖细胞在位于两个性腺臂的远端区域的两个成体干细胞龛中产生（Hansen和Schedl，2013; Kimble和Seidel，2013）。在野生型雌雄同体中，有丝分裂生殖细胞仅限于性腺臂的远端，干细胞生态位。野生型生殖细胞是合胞体，并且包含延伸通过性腺臂中心区域的共同细胞质的开口。 ℃。线虫肿瘤种系突变体在整个性腺中增加了生殖细胞的有丝分裂增殖。我们发现肿瘤种系突变体通常具有完整的细胞生殖细胞，其含有完整的质膜（Chaudhari等人，2016）。这种细胞化允许生殖细胞的分离及其在培养中的维持。该方案描述了分离和维持培养物中肿瘤突变体的生殖细胞的方法学和组织培养基。虽然已经描述了用于C的主要培养物的培养基。 elegans 胚胎和幼虫细胞（Strange等人，2007; Zhang和Kuhn，2013），生殖细胞在这种培养基中不能存活（Chaudhari等人。 ...
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| Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay
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Author:
Date:
2017-04-05
[Abstract] An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous ...
[摘要] 免疫反应可以被致病性刺激以及内源性危险信号激活，引发模式识别受体的激活并引发导致炎症的信号级联。该方法使用THP1-Blue TM 细胞，其是含有能够检测NF-κB诱导的转录激活的胚胎碱性磷酸酶报告基因的人单核细胞系。通过两种不同的激动剂刺激toll样受体4（TLR4）后，通过评估NF-κB活化来验证该方案：从革兰氏阴性细菌的细胞壁衍生的脂多糖（LPS）和腱生蛋白C，细胞外基质蛋白其组织损伤引起其表达。然后我们使用该方案筛选潜在的新的内源性TLR4激动剂，但是该方法也可以用作快速，经济和可靠的方法来测定导致TLR依赖性NF-κB活化的其它炎性刺激的活性。
免疫系统已经发展成不仅识别病原性刺激物，例如细菌成分和病毒核酸，还包括内源性危险信号，包括从坏死细胞分泌的蛋白质或表达于组织损伤。通过模式识别受体感测到两种类型的刺激，启动触发炎症反应的信号级联。活化的B细胞（NF-κB）的核因子κ-轻链增强子是激活对感染和组织损伤的免疫应答所必需的转录因子。 NF-κB在模式识别受体激活后，在广泛的炎症介质的表达中具有重要作用，包括细胞因子（如肿瘤坏死因子α[TNFα]，白细胞介素-6和白细胞介素-1），趋化因子例如白细胞介素-8或CXCL1），蛋白酶，生长因子和MHC相关分子等。为了评估在TLR4下游的该途径的活化，我们使用市售的细胞系THP1-Blue ...
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| IFN-α/β Detection Assay Using Sensor Cell Lines
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Author:
Date:
2014-05-20
[Abstract] Type I interferons (IFN-α/β) play an important role in host resistance to viral infections. Signaling through the JAK-STAT pathway, IFN-α/β stimulates response elements (ISRE) in the promoters of ISG to regulate their expression (reviewed in Reference 2). This method was adapted from InvivoGen to specifically detect and quantify IFN-α/β secreted in response to virus infection. HEK-Blue™ IFN-α/β cells were generated by stably introducing the human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. The activation of this pathway is made detectable by the addition of a reporter gene expressing a secreted embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG activated through an ISRE-dependent ...
[摘要] I型干扰素（IFN-α/β）在宿主对病毒感染的抗性中起重要作用。 通过JAK-STAT途径的信号传导，IFN-α/β刺激ISG启动子中的反应元件（ISRE）以调节它们的表达（参见参考文献2）。 此方法改编自InvivoGen以特异性检测和量化响应于病毒感染分泌的IFN-α/β。 通过将人STAT2和IRF9基因稳定地引入HEK293细胞中以产生完全活性的I型IFN信号传导途径，产生HEK-Blue TM IFN-α/β细胞。 通过加入在ISG54启动子控制下表达分泌性胚胎碱性磷酸酶（SEAP）的报告基因，可以检测该途径的活化。 ISG54是通过I型IFN的ISRE依赖性机制激活的公知的ISG
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