{{'Search' | translate}}
 

50 ml and 15 ml Conical centrifuge tubes

Falcon ® 15mL高清PP离心管,圆锥底,带圆顶密封螺帽

Company: Corning
Catalog#: 352096
Bio-protocol()
Company-protocol()
Other protocol()

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
Author:
Date:
2017-10-05
[Abstract]  Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the ... [摘要]  溶酶体是具有降解递送到这些隔室的货物的活性水解酶所需的酸性环境的内吞途径的末端。 这种内溶溶酶体的货物递送和降解过程是一个严格调节的过程,对于维持细胞体内平衡是重要的。 Cargos如EGF(表皮生长因子),Dil-LDL(3,3'-二十八碳基碳代花青 - 低密度脂蛋白),葡聚糖,DQ-BSA(染料淬灭 - 牛血清白蛋白)等常规 由研究人员用来分析各种蛋白质在内吞途径中的作用。 在缺乏或过表达感兴趣的基因的细胞中的DQ-BSA的贩运是用于鉴定各种蛋白质在内吞运输途径中的作用的有用测定。 该方案描述了可用于研究各种细胞类型的吞噬运输的DQ-Red BSA运输测定。
【背景】细胞不断地与其细胞外环境交换物质,并且在这个过程中,它们将货物内部化在质膜的囊泡中。这种内部货物被运送到早期的内体,从那里它可以通过再循环内体回到质膜或进入规范的内吞途径。一旦被注定要退化,货物就会移动到后期的内体,并最终与溶酶体融合,其中活性水解酶消化货物(Jovic等人,2010)。这些内吞室具有其内腔的特征pH。早期内体体内的pH值范围为5.9-6.8,晚期内体的pH范围为4.9-6.0,溶酶体最为酸性,pH范围为4.5-5.0(Maxfield和Yamashiro,1987)。溶酶体的酸性环境对于存在于其管腔中的水解酶和货物降解的活性是必需的(Garg等人,2011; ...

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
Author:
Date:
2017-10-05
[Abstract]  The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal ... [摘要]  从mRNA分子产生蛋白质的效率可以在转录本,细胞类型和细胞状态之间广泛变化。准确测定mRNA翻译效率的方法对获得对转录后基因调控的机理理解至关重要。测量翻译效率的一种方法是确定与mRNA分子相关的核糖体的数目,归一化为编码序列的长度。分析单个mRNA的主要方法是蔗糖梯度分级,其基于结合核糖体的数目物理分离mRNA。在这里,我们描述了精确分析与核糖体的mRNA相关性的简化方案。与以前的方案相比,我们的方法结合内部控制和改进的缓冲条件,共同减少由非特异性mRNA - 核糖体相互作用引起的伪像。此外,我们的直接分数qRT-PCR方案消除了从梯度部分中RNA纯化的需要,这大大减少了所需的手动时间量,并促进了多个条件或基因靶标的并行分析。此外,在该过程中不产生苯酚废物。我们最初开发了协议来研究S-HAC1 mRNA的翻译抑制状态。但是我们还详细介绍了哺乳动物细胞系和组织的适应程序。
【背景】将mRNA翻译成蛋白质是一种高度调节的过程,其可以以不同的速率发生,这取决于基因,细胞环境或环境。翻译起始,延伸和终止的每个步骤可以是最终影响与mRNA相关的核糖体数量的调节点(Dever和Green,2012; ...

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
Author:
Date:
2017-09-20
[Abstract]  This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and ... [摘要]  该方案旨在标准化海马和脑皮质神经元原代培养物的体外实验。 培养物应从胚胎发育期间分离的细胞制备,当神经元前体细胞尚未完全分化时。 这有助于提高细胞的质量和数量,同时提供通常在分化的神经元解离期间发生的最小的细胞死亡。 在合适的条件下,在培养皿或多孔板中铺板的细胞将随着时间的推移而发展和建立突触接触,因为神经元培养基提供分化所需的营养和营养因子。 在这个协议中,我们描述了制备皮质和海马神经元培养物的方法。
【背景】本方案描述了使用补充有NeuroCultTM SM1的Neurobasal培养基(Chen等人,2008)的大鼠海马和脑皮层神经元的原代培养物的制备。 NeuroCultTM SM1的组成基于B27补充剂的制剂(Brewer等,1993),但是前者的混合物被发现提高了神经元培养的质量,部分地通过用全转运蛋白替代载脂蛋白转运蛋白 Chen et al。,2008)。 此外,NeuroCultTM SM1的化学成分在原始出版物中有更详细的描述,可以更好地控制实验条件。 用化学确定的培养基制备的神经元培养物的特征在于存在低百分比的星形胶质细胞。 通过添加有丝分裂5-氟-2'-脱氧尿苷的化学抑制剂可以防止维持更长时间的培养物中星形胶质细胞的增殖以允许神经元分化。

Comments