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Dulbecco's Modification of Eagle's Medium (DMEM)

Dulbecco''s Modification of Eagle''s Medium(DMEM)

Company: Mediatech
Catalog#: 10-013-CV
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A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
Author:
Date:
2017-08-20
[Abstract]  Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, ... [摘要]  原代神经元是鉴定用于治疗神经变性疾病的治疗性寡核苷酸的理想细胞系统。然而,由于原代细胞的敏感性,使用经典方法转染小干扰RNA(siRNA)是费力的,并且通常显示低效率。寡核苷酸化学的最新进展使得稳定和疏水修饰的小干扰RNA(hsiRNA)的发展成为可能。这种新型的寡核苷酸治疗剂显示出非常有效的自我传递性质,并且在体外和体内支持有效和持久的效果。我们开发了高通量的体外测定法来鉴定和测试原代神经元培养物中的hsiRNA。为了简单,快速,准确地量化数百个hsiRNA的mRNA沉默,我们使用QuantiGene 2.0定量基因表达测定法。这种高通量,96孔板测定法可以直接从样品裂解液中定量mRNA水平。在这里,我们描述了一种制备96孔板格式的小鼠原代皮质神经元的短期培养物用于寡核苷酸治疗剂的高通量测试的方法。该方法支持在短短两周内测试hsiRNA文库和鉴定潜在的治疗方法。我们详细介绍了从初级神经元准备到数据分析的高通量测定工作流程的方法。该方法可以帮助鉴定用于治疗各种神经疾病的寡核苷酸治疗剂。
【背景】寡核苷酸治疗剂代表了通过沉默突变蛋白的表达,可以靶向任何遗传定义的病症的新一类药物。具体地,siRNA是负载于RNA诱导的沉默复合体(RISC)中的双链寡核苷酸,并且可以在mRNA翻译之前使mRNA沉默。然而,未修饰的siRNA是不稳定的,并且不能在没有阳离子脂质制剂的帮助下进入细胞,其可能对原代细胞如神经元有毒性。在本协议中,我们使用自我递送,疏水修饰的siRNA(hsiRNA)进行mRNA沉默。最近在寡核苷酸化学方面的进展使得这些稳定的hsiRNA的设计促进了细胞内化,有效进入RISC以及有力击倒靶基因(Byrne等,2013; ...

Mouse Müller Cell Isolation and Culture
Author:
Date:
2017-08-05
[Abstract]  Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells. [摘要]  Müller细胞是视网膜上的主要支持和保护性胶质细胞。 与鱼类不同,它们已经失去了在哺乳动物中再生视网膜的能力。 但是,哺乳动物Müller细胞仍然保留了具有不同程度的自我更新和分化潜能的某些视网膜干细胞特性,从而在基于细胞的疗法中治疗视网膜变性疾病具有优点。 在我们的实验室,我们使用酶法来分离,纯化和培养小鼠Müller细胞。
【背景】Müller胶质细胞是视网膜的主要谱系,其功能是通过神经营养因子的合成,神经递质的摄取和再循环,离子的空间缓冲和血液视网膜屏障的维持来维持视网膜稳态(Bringmann等人,,2006; De Melo Reis等人,2008)。 Müller神经胶质细胞作为鱼类中的视网膜祖细胞和干细胞,在有限的范围内作为鸟类(Vihtelic和Hyde,2000; Fischer and Reh,2001)。但是,哺乳动物Müller细胞已经失去了再生视网膜的能力,尽管仍然保留成体干细胞的某些性质,例如视网膜损伤时的增殖。研究恢复哺乳动物Müller细胞丧失能力以修复视网膜损伤和理解底层机制的研究在与模型动物视网膜分离的原代细胞的实验室进行。蛋白水解酶广泛用于Müller细胞解离,木瓜蛋白酶的损伤较小,比其他蛋白酶更有效。 ...

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
Author:
Date:
2017-06-05
[Abstract]  The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of ... [摘要]  消除HIV-1患者的主要障碍是后整合延迟(Finzi等人,1999)。抗逆转录病毒治疗仅针对主动复制病毒,而具有低转录活性或无转录活性的潜伏感染仍未得到治疗(Sedaghat等人,2007)。为了消除病毒性水库,一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期(Deeks,2012)。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制,并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近,许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端(BET)家族的成员的治疗潜力(Filippakopoulos等人,2010; Dawson等人& / em>,2011; Delmore等人,2011),其包括BRD2,BRB3,BRD4和BRDT。小分子BET抑制剂,例如JQ1(Filippakopoulos et al。,2010; Delmore等人,2011),I-BET(Nicodeme等人< / ...

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