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1x Dulbecco’s phosphate buffered saline

DPBS,无钙,无镁

Company: Thermo Fisher Scientific
Catalog#: 14190250
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In vivo Mouse Mammary Gland Formation
Author:
Date:
2020-07-05
[Abstract]  For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several In vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of “cleared” recipients and mammary cells for implantation, the surgery process and ... [摘要]  [摘要 ] 多年来,乳腺一直是研究成体干细胞的自我更新和分化以及这些过程的调控机制的完美例证。为了评估给定的基因和/或其他因素对乳腺细胞的干性,有几个函数体外测定法开发出来,如微球体由mRNA表达形成试验,干细胞标志物的检测或流式细胞术等。然而,在雌性小鼠清除的脂肪垫中整个坐骨的重建能力是估计细胞干性的黄金标准。在这里,我们描述了体内的分步操作方案 乳腺形成测定,包括准备“清除”的受体和用于植入的乳腺细胞,手术过程以及如何评估实验结果。结合通过基因编辑和/或药物处理对乳腺细胞的操作,该方案在乳腺干细胞和乳腺发育的研究中可能非常有用。

[背景 ] 作为哺乳动物最典型的器官之一,乳腺(MG)是外分泌腺,负责泌乳。MG的发育受某些性激素的控制,这些激素的水平精确地调节了MG在不同发育阶段的结构,细胞组成和功能变化(Henigighausen and Robinson,2005)。许多遗传和环境因素都参与了乳腺干细胞的调控和MG的发育。为了研究这些因素的功能和机理,已经开发了几种方法,特别是用于评估乳腺细胞的干性。先前的研究表明,只有MG的基底细胞而非管腔细胞能够在受体雌性小鼠清除的脂肪垫中重建上皮树,这表明乳腺干细胞仅存在于基底谱系中(Van Keymeulen 等,2011)。 )。后来,包括我们在内的许多研究发现了乳腺干细胞的几种标志物(Prater ...

Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
Author:
Date:
2020-06-20
[Abstract]  Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these ... [摘要]  [摘要]核细胞质运输缺陷被认为在神经退行性疾病中发挥作用,包括肌萎缩性侧索硬化症(ALS)。鉴于这一过程的重要性和复杂性,了解这些畸变何时发生以及涉及哪些途径是非常重要的。在这里,我们利用CRISPR-Cas9技术来设计细胞系,稳定地表达荧光素蛋白在细胞核和细胞质之间穿梭的选择的卡里菲林。为了验证这个协议,我们测量了一个ALS相关的核细胞质运输途径,在疾病相关的多肽poly-PR的存在。这种技术允许测量在神经退行性疾病相关的背景下,在完整细胞中的一个特定的活性核细胞质运输途径。此外,这些实验可以在不需要昂贵的设备的情况下进行,并有可能升级为高通量筛选目的。

[背景]核细胞质运输对细胞稳态至关重要,并通过形成水通道的大型多蛋白复合物穿过核膜,称为核孔复合物(NPCs)(Stoffler et al., 1999; Ryan and Wente, 2000)。虽然这些通道允许小蛋白被动地平衡穿过核膜(被动运输),但大多数蛋白质似乎是由核素主动运输的,即进口蛋白或出口蛋白(主动运输)。含有核定位信号(NLS)的货物蛋白被导入素所定位。已经确定了各种受体介导的导入途径,但最有特色的途径涉及导入素-β1/导入素-α(KPNB1/KPNAx),其中货蛋白含有经典的NLS(cNLS),以SV40大T抗原NLS为例(Lange et al., ...

Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
Author:
Date:
2018-06-20
[Abstract]  Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular ... [摘要]  双链RNA是一种有效的病原体相关分子模式(PAMP),作为病毒复制的副产物和病毒感染的众所周知的标志产生。 病毒dsRNA可以从感染的细胞释放到细胞外空间并通过胞吞作用被邻近细胞内化。 哺乳动物具有能够检测导致产生I型干扰素(IFN)的病毒dsRNA(例如内体Toll样受体3(TLR3)和胞质RIG-1样受体(RLR))的多模式识别受体(PRR)。 因此,病毒dsRNA的细胞内定位可以提供对导致先天免疫激活的下游信号传导途径的了解。 在这里,我们描述了一种测量细胞外dsRNA摄取和分别通过流式细胞仪和共聚焦显微镜观察内化dsRNA的亚细胞定位的定量方法。

【背景】双链RNA(dsRNA)是病毒复制的常见副产物,通过产生I型干扰素(IFN)和其他促炎细胞因子(Nellimarla和Mossman,2014)是抗病毒免疫的有效激活剂。病毒的dsRNA通过TLR3核内体内所感测(松本等人,2003年)或在由RIG-I样受体(RLRS),RIG-I和MDA-5(加藤等人,2006)。在裂解感染,这些dsRNA可以被释放到细胞外空间,它们结合于相邻小区,如A类清道夫受体(SR-A)和RAFTLIN表面受体,并且随后经由网格蛋白介导的内吞作用内在化(伊藤制 2008; DeWitte-Orr等人,2010; Watanabe等人,2011; Dansako等人,, ...

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