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Tissue culture dish (35 mm)

Company: BD
Catalog#: 353001
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Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
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Date:
2018-07-05
[Abstract]  Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure ... [摘要]  来自产后啮齿动物的小脑颗粒神经元(CGN)已被广泛用作研究神经元发育,生理学和病理学的模型。 CGN体外培养维持成熟小脑颗粒细胞在体内显示的相同特征,包括发育致密的神经炎网络,神经元活动,神经递质释放和神经元的表达 蛋白质标记。 此外,CGN代表了梭菌神经毒素(CNT)研究的便利模型,最着名的是破伤风和肉毒杆菌神经毒素,因为它们大量表达CNT受体和神经元内基质, ie ,可溶性N-乙基马来酰亚胺 - 敏感因子激活蛋白受体(SNARE蛋白)。 在这里,我们描述了从出生后大鼠/小鼠获得高纯度CGN培养物的方案和用CNT中毒的简便方法。 我们还说明了评估CNT活性及其抑制的方便方法。

【背景】梭菌神经毒素(CNT)的大家族由破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNT)的多种变体形成,它们分别是破伤风和肉毒中毒的神经麻痹毒素(Schiavo et al。,2000; Johnson和Montecucco,2008; Rossetto et al。,2014)。 TeNT,七种BoNT血清型(BoNT / A至/ G)及其许多亚型是金属蛋白酶,通过切割SNARE蛋白(可溶性N-乙基马来酰亚胺敏感因子激活蛋白受体),三种必需蛋白质来阻断神经递质的释放而引起神经麻痹。控制突触小泡与突触前质膜的融合(Rossetto et al。,2014; ...

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
Author:
Date:
2018-05-05
[Abstract]  Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution ... [摘要]  骨髓造血干细胞(HSC)需要骨髓微环境来维持和增殖。 骨髓间充质基质细胞(MSC)的培养为体外HSC存活提供适当的环境信号。 在这里,我们提供了描述MSCs培养条件的详细方案,从小鼠骨髓中流式细胞术分离HSCs,并进行称为鹅卵石区域形成细胞(CAFC)分析的MSC和HSC的共培养。 总而言之,CAFC分析可用作高通量体外筛选模型,其中努力了解和开发复杂骨髓疾病的治疗方法。 该方案需要培养MSC,分离LSK细胞(HSC)和执行有限稀释CAFC测定3至4周。

【背景】HSC的增殖,存活和分化潜力非常依赖于其微环境,也被称为小生境。骨髓MSC支持HSC以使其在骨髓龛中保持静止状态。由生态位接收的内在和外在信号有助于将HSC分化为也称为造血的成熟血细胞谱系,而不诱导异常扩增(Yoshihara等人,2007; Spindler等人 ,2014; Hu等人,2016)。鹅卵石区域形成细胞试验(CAFC试验)是长期骨髓HSC和MSC的体外共培养试验。当培养MSC在组织培养皿中完成融合时,将HSC铺在MSC上(de Haan和Ploemacher,2002)。 CAFC测定与骨髓的体内研究相当,并且可用作快速筛选测定以测试HSC的干细胞活性和MSC的支持活性(Ploemacher等人, ...

Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines
Author:
Date:
2018-03-20
[Abstract]  The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells. [摘要]  主要纤毛是一种非运动感觉细胞器,其装配和拆卸与细胞周期进程密切相关。 初级纤毛在静止细胞中从基体拉长并随着细胞重新进入细胞周期而被吸收。 睫状动力失调与纤毛病和其他人类疾病有关。 体外血清刺激的睫状体装配/分解测定已经在解决睫状动力学中感兴趣的蛋白质的功能方面受到欢迎。 在这里,我们描述了转染人视网膜色素上皮细胞(RPE-1)和对转染细胞进行睫状体装配/分解测定的充分测试的方案。

【背景】初级纤毛是毛发样感觉细胞器,其在G 0 / G 1期出现,并且在细胞周期的S期之前分解(Tucker等, et al。,1979)。先前的研究已经证实,某些未转化的细胞类型(即,甚至是RPE-1细胞,3T3成纤维细胞和小鼠胚胎成纤维细胞[MEFs])可以被饿死以诱导静止和睫状体形成。随后的血清再次添加触发双相睫状体吸收,其在刺激后2小时和24小时达到峰值(Tucker等人,1979; Li等人,2011) 。该现象为文献中常用的血清刺激的睫状体组装/分解测定奠定了基础,以鉴定参与睫状体组装和拆卸的蛋白质(Pugacheva等人,2007; ...

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