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Tissue culture microscope

反向常规显微镜

Company: Nikon
Catalog#: Eclipse TS100
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Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification
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Date:
2017-12-20
[Abstract]  The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of guard cell protoplasts and measure H+-pumping in the protoplasts. It is also necessary to determine the protein amount of H+-ATPase. In this protocol, we describe the procedures required for these preparations and measurements. [摘要]  响应蓝光的植物气孔的开放是由保卫细胞中的质膜H + -ATPase驱动的。 为了评价体内H + -ATP酶的激活,我们可以使用H + +保卫细胞对蓝光的响应,fusicoccin。 为此,需要制备大量的保卫细胞原生质体,并测量原生质体中的H + - 抽吸。 还需要确定H + -ATP酶的蛋白质量。 在这个协议中,我们描述了这些准备和测量所需的程序。

【背景】响应于蓝光的气孔的开放是由穿过保卫细胞质膜上的H +介导的膜超极化驱动的(Assmann等,1985; Shimazaki等人,1986),并且是由质膜H + -ATP酶引起的(Kinoshita和Shimazaki,1999)。 H + -ATP酶在膜上产生电化学梯度,并提供植物细胞中许多次级运输所需的能量。然而,测量体内H + -ATP酶活性并不容易。利用保卫细胞的蓝光敏感特性,我们的方法可以将体内H +泵送作为体内测量H + + 使用拟南芥保卫细胞原生质体的ATP酶活性(Ueno等人,2005)。与通过蛋白质印迹(Yamauchi等人,2016)的Hβ+ -ATPase定量一起,该方法允许比较Hβ+ -ATPase活性不同的条件或突变背景。

Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells
Author:
Date:
2017-12-05
[Abstract]  Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes ... [摘要]  外来体已经成为细胞间通讯的重要介质。 它们存在于细胞外环境中,因此可以被邻近或远处的细胞轻易获得。 它们在囊泡内携带mRNA,microRNA和蛋白质,并一度被受体细胞内化; 它们可以通过诱导抗病毒状态向疾病进展调节多种信号传导途径,具有多效性。 我们以前已经显示,携带基因组长度复制子的丙型肝炎病毒(HCV)感染的肝细胞或肝癌细胞在培养上清液中分泌外泌体。 这些外来体被肝星状细胞(HSC)摄取,并在HCV感染期间激活它们以诱导纤维化。 在这里,我们描述了外来体分离和肝星状细胞摄取BODIPY标记的外来体的详细的协议。

【背景】外来体是直径为40-150nm的小的膜结合的细胞外囊泡。已在所有体液中鉴定出它们,包括尿,羊水,血清,唾液,母乳,脑脊液,鼻分泌物和组织培养的细胞系的上清液中。外泌体通过转移微小RNA(mRNA),mRNA和蛋白质来介导细胞与细胞之间的通讯。 其可被邻近或远处的细胞摄取,并随后促进受体细胞中的信号传导(Schorey等人,2015)。外泌体富含蛋白质,包括四跨膜蛋白家族成员(CD9,CD81,CD63),热休克蛋白(Hsp60,Hsp70,Hsp90)和多泡体蛋白(annexins,RabGTPases和内体分选复合物,蛋白质)。最近的研究表明,外来体可以影响免疫应答(Li等人,2013; ...

In vitro Homeostatic Proliferation of Human CD8 T Cells
Author:
Date:
2017-11-20
[Abstract]  Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing maintenance of effector functions in long-lived memory T cells during antigen-independent proliferation, human naïve and memory CD8 T cells can be sorted from peripheral blood mononuclear cells (PBMCs), labeled with the proliferation-tracking dye carboxyfluorescein succinimidyl ester (CFSE), and then purified based on their levels of cell division. This allows investigators to assess differences in the desired molecular target in cells that have ... [摘要]  长寿T细胞介导的免疫需要在无抗原环境中持续存在记忆T细胞,同时还保持提高的回忆效应器功能的能力。这种不依赖于抗原的体内平衡增殖部分由常见的γ链细胞因子IL-7和IL-15介导。为了进一步探索在抗原非依赖性增殖过程中维持长效记忆T细胞中效应子功能的机制,可以从外周血单核细胞(PBMCs)中分选人幼稚和记忆CD8T细胞,用增殖跟踪染料羧基荧光素琥珀酰亚胺酯(CFSE),然后根据它们的细胞分裂水平进行纯化。这使得研究人员能够评估经历细胞因子驱动的增殖的细胞中期望的分子靶点的差异。我们在这里提供了一个协议,用于评估在分裂和未分裂的人类幼稚和记忆CD8 T细胞中的表观遗传程序,在用IL-7和IL-15培养7天之后,以说明这种方法如何阐明控制在长寿记忆CD8T细胞的稳态期间保持效应子功能。
【背景】获得性免疫的一个主要特征是对先前遇到的病原体的免疫记忆的发展(Plotkin等人,2013)。记忆CD8 T细胞在为宿主以前遇到的病原体提供终生保护方面发挥重要作用,但为了提供长寿命的保护,T细胞必须已经获得了在无抗原中持续并保持效应功能的能力环境。在此期间,记忆T细胞响应于IL-7/15细胞因子进行抗原非依赖性增殖,尽管程度不同。

记忆CD8 T细胞的总池是几种不同的细胞亚群的异质组合,其对稳态细胞因子的反应不同。例如,CD8 ...

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