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Fetal Bovine Serum

胎牛血清(FBS)

Company: Thermo Fisher Scientific
Catalog#: 26140079
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3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
Author:
Date:
2018-06-05
[Abstract]  Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, ... [摘要]  细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而,在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用,并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性(Edmondson等人,2014)。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型(肿瘤球体)在该领域已经获得了越来越多的普及(Weiswald等人,2015)。使用基质嵌入或封装的球体,悬滴培养的球体,磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中,我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样,该方案也可用于直接从肿瘤组织中分离3D癌细胞系。

【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法(Pampaloni等人,2007; ...

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; ...

Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons
Author:
Date:
2018-01-20
[Abstract]  Release and recycling of synaptic vesicles are essential for neurotransmission and synaptic plasticity. To gain mechanistic understanding of these processes, direct measurements of vesicle release and retrieval is indispensable. Styryl dyes like FM1-43 and FM4-64 have been widely used for this purpose and their loading and unloading are reliable measurements for synaptic vesicle release and retrieval in cultured neurons. This protocol describes in detail the procedure of using styryl dyes to label and measure synaptic vesicle uptake and release in cultured rat hippocampal neurons. We also include a brief description of hippocampal culture. In the end, we briefly discuss the commonality and difference among FM dye, pH-sensitive fluorescent proteins and quantum dots in terms of measuring ... [摘要]  突触小泡的释放和再循环对于神经传递和突触可塑性是至关重要的。 为了获得对这些过程的机械理解,直接测量囊泡释放和回收是必不可少的。 苯乙烯基染料如FM1-43和FM4-64已被广泛用于此目的,其装载和卸载是可靠的测量突触小泡释放和恢复培养的神经元。 该协议详细描述了使用苯乙烯基染料来标记和测量培养的大鼠海马神经元中的突触小泡摄取和释放的程序。 我们还包括对海马文化的简要描述。 最后,我们简要讨论FM染料,pH敏感荧光蛋白和量子点在测量突触小泡行为方面的共性和差异。
【背景】突触小泡是神经传递不可或缺的,因为它们是化学突触中负责神经递质释放的唯一细胞器。它们的数量,释放概率,融合动力学和再循环路线定义了突触传递和神经元交流。已经开发了多种用于探测突触囊泡的工具,包括突触后神经元的电生理学记录,膜运输的电容测量,可氧化的发射体的电流分析,固定突触的电子显微镜成像以及活神经元中的囊泡标记的荧光成像。在所有现有的方法中,最后一个是不仅产生关于个体突触的空间和时间信息,而且提供高吞吐量(即,来自不同神经元的单个突触的更多数据点)的唯一方法。已经开发了基于不同定向和报告机制的各种荧光探针。二十多年前发明的苯乙烯基染料(即FM染料,包括FM1-43,FM4-64,FM5-95)仍然是一种可靠而方便的工具。由于其对脂质膜的中等亲和力及其对脂质敏感的排放,可以容易地装载到再循环的突触囊泡中并且当这些囊泡被胞吐出时释放。使用更敏感的光电探测器如EMCCD,FM染料可以报告单个囊泡释放事件。在这里,我们提供了一个相对完整的基于FM的啮齿动物海马神经元原代培养的突触小泡释放成像的描述。此外,我们还讨论了FM染料和其他荧光泡标签的共性和区别。 ...

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