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Fetal Bovine Serum

胎牛血清

Company: Sigma-Aldrich
Catalog#: F7524
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An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist
Author:
Date:
2018-07-05
[Abstract]  One fundamental property of the TNR receptor (TNFR) family relates to how ‘signal quality’ (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., ... [摘要]  TNR受体(TNFR)家族的一个基本特性涉及“信号质量”(受体连接或交联的程度)如何影响受体连接的结果,例如肿瘤细胞中的死亡诱导。毫无疑问,膜呈递配体(通过与表达配体的第三方细胞共培养通过细胞表面呈递递送至靶细胞)在体外诱导更大程度的癌细胞死亡非交联激动剂(激动性抗体和/或重组配体)。 CD40受体集中体现了TNF受体 - 配体相互作用的这种基本特性,因为CD40交联的程度决定了细胞命运。膜呈递CD40配体(mCD40L),但不是可溶性激动剂(例如,激动性抗CD40抗体),诱导高水平的促炎细胞因子分泌并导致恶性肿瘤细胞广泛死亡(细胞凋亡)但不是正常的)上皮细胞。在本文中,我们描述了通过mCD40L激活CD40并随后检测细胞凋亡的各种特征(包括细胞膜透化,DNA片段化,半胱天冬酶活化)以及细胞内细胞死亡介质检测的共培养系统(包括衔接蛋白,促凋亡激酶和活性氧,ROS)。

【背景】TNFR及其配体在调节淋巴组织以及上皮(尤其是癌)细胞中的细胞增殖或死亡中的作用已经在广泛研究中,因为它们诱导细胞死亡(主要通过细胞凋亡)的能力代表了有希望的目标。用于癌症治疗。然而,重要的是,当以可溶性对膜结合形式存在时,TNFR激动剂引发细胞死亡的能力存在明显差异。当作为单独治疗施用时,可溶性激动剂通常表现出相对低的细胞毒性效力,而膜呈递的配体似乎是优越的(Albarbar ...

An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells
Author:
Date:
2018-05-05
[Abstract]  In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death. [摘要]  在该协议中,我们描述了通过碘化丙啶(PI)染色的贴壁哺乳动物细胞的活细胞成像来监测细胞增殖和死亡的方法。 PI广泛用于评估细胞死亡。 但是,它通常用于终点检测。 最近,我们通过自动成像实现了PI的实时细胞死亡评估。 将细胞以96孔形式接种,并且在附着后,将处理与PI一起直接加入到孔中。 之后,通过计算机软件对细胞进行自动延时成像和定量。 相衬和荧光图像的组合分析允许评估对细胞增殖的处理效果以及细胞死亡的程度和动力学。

【背景】多种基于细胞的测定可用于确定细胞死亡,但其中大多数测定包括MTT(溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓)测定,晶蓝染色,和各种基于流式细胞术的方法,都有作为终点分析的限制。最近,我们使用自动荧光成像系统(Essen Bioscience的IncuCyte ZOOM)(Sehgal等人,2017)将碘化丙啶(PI)用于细胞死亡的活细胞评估。通过对相差图像进行组合分析,可以同时检测细胞抑制和细胞毒性效应。这种方法证明是可靠和可重复的,以及非常简单和便宜。在我们最近的出版物中,我们将它用于三种不同的癌细胞系(LNCaP,PC3和MCF7),以有效地确定和比较ER Ca 2 +泵抑制剂毒胡萝卜素各种药物类似物(Tg )(Sehgal et ...

The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
Author:
Date:
2018-05-05
[Abstract]  Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw ... [摘要]  自噬是维持真核生物细胞稳态的关键因素,包括癌症和神经退行性疾病在内的许多疾病都与自噬的改变有关。研究自噬的兴趣在过去的二十年里急剧增长,并且还有用于研究这种高度动态和复杂过程的方法库。通常通过Western印迹和各种显微镜技术来评估自噬相关蛋白的表达和/或定位中的变化。这样的分析可能表明自噬相关过程的改变,并且关于正在研究的特定标记物的信息。然而,由于这些蛋白质是自噬机制的一部分,而不是自噬性货物,所以它们不能用于得出关于自噬载货流量的结论。在这里,我们提供了一个协议,通过使用长寿命的蛋白质降解测定来定量评估体积自噬流量。我们的程序追踪14 C缬氨酸标记的蛋白质的降解是简单和快速的,允许并行处理相对大量的样品,并且原则上可以与任何贴壁细胞一起使用线。最重要的是,它可以通过自噬途径定量测量内源货物流量。因此,它是研究自噬活动的黄金标准之一。

【背景】脉冲追踪标记方法已用于研究蛋白质周转数十年。在此处描述的长寿命蛋白质降解(LLPD)测定中,培养细胞的蛋白质组用14 ...

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