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0.4% Trypan blue

台盼蓝溶液

Company: Sigma-Aldrich
Catalog#: 93595
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Neutrophil Extracellular Trap Killing Assay of Candida albicans
Author:
Date:
2020-08-20
[Abstract]   Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires ... [摘要]  [摘要 ] 丰人病原体念珠菌白色念珠菌是顶级领先的原因之一全球总卫生保健相关血流感染。中性粒细胞是清除白色念珠菌感染的主要效应细胞。我们的研究表明,小鼠中性粒细胞利用两种独立的机制杀死白念珠菌:一种是CR3下游NADPH氧化酶依赖性机制,它可以杀死调理过的白色念珠菌。另一个是dectin-2介导的NADPH氧化酶非依赖性中性粒细胞胞外捕获物(NET),它杀死未调理的白色念珠菌。中性粒细胞杀死调理过的白色念珠菌 需要吞噬生物体并产生活性氧(ROS)。大多数现有的协议的测定中性粒细胞杀死白色念珠菌需要使嗜中性粒细胞吞噬生物体后洗涤步骤。根据定义,NET在细胞外杀死生物。因此,重要的是要跳过洗涤步骤,并向孔中添加最佳比例的嗜中性白细胞和白色念珠菌。为了证明NET的作用,有必要比较用微球菌核酸酶(MNase )(一种消化NET的酶)处理的嗜中性粒细胞的杀伤能力与用热灭活的MNase 处理的嗜中性粒细胞的杀伤能力。MNase 还用于释放与NET绑定的真菌元素以进行计数。该协议可用于测定其他生物膜形成生物的NET杀灭。

[背景 ] ...

Generation of Functional Mouse Hippocampal Neurons
Author:
Date:
2020-08-05
[Abstract]  Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos’ isolation, culturing conditions and ... [摘要]  [摘要] 原代培养小鼠海马神经元是一种非常有用的体外模型用于研究神经元的发育,轴突和树突的形态,突触功能,以及许多其他神经元的特征。这里我们描述了从小鼠胚胎海马(E17.5/E18.5)产生初级神经元的一步一步的过程。根据不同的实验目的,用该方法产生的海马神经元可以在不同的组织培养皿中进行培养,并能在不到一周的时间内产生一个可靠的来源。该方案涵盖了神经元培养物的制备、培养和鉴定的所有必要步骤,包括解剖器械的说明、胚胎分离的手术程序、培养条件以及培养物纯度和分化的评估。通过分析培养6天时的钙显像动力学来评估神经元的活性。

[背景] 海马体是一个非常典型的大脑结构,对重要的大脑功能如记忆、空间导航、情绪记忆和学习至关重要。从解剖学上讲,小鼠海马体有一个清晰的C形结构,很容易定位和分离。在细胞水平上,它主要由锥体细胞组成,与其他脑区相比,中间神经元和胶质细胞较少(Kaech和Banker,2006)。因此,海马体是从野生型或基因工程小鼠模型中产生高纯度原代神经元培养物的理想区域,可用于疾病建模或研究神经元功能的多个方面,如突触传递和电生理特性、对神经毒性的敏感性,分化与衰老(;;;;)。Busche,2018Koyama和Ikegaya,2018Molnar,2011Wu等人,2019Rush等人,2020年

已经制定了许多协议,通过与神经胶质喂食器共同培养神经元来产生皮层和海马神经元(Kaech和Banker,2006),描述了用水凝胶微纤维封装的星形胶质细胞的三维神经元培养系统(Kim等人,2020年),长期向培养基中补充生长因子神经元培养(Ray ...

Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
Author:
Date:
2017-01-20
[Abstract]  Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates the Ficoll solution from the blood to be drawn. Once blood is drawn into CPTs, they can be centrifuged to separate the PBMC, then shipped (if desired) to a processing lab. The processing lab removes the PBMC from the upper compartment of the tube (above the gel plug), washes the PBMC, and can cryopreserve them using DMSO-containing media, as detailed in this protocol. [摘要]  外周血单核细胞(PBMC)分离通常通过Ficoll-Hypaque密度梯度离心来进行,Ficoll-Hypaque是一种劳动密集型程序,需要熟练的技术人员并且可以有助于样品变异性。细胞制备管(CPT)是含有Ficoll-Hypaque的Vacutainer采血管和将Ficoll溶液与待吸取血液分离的凝胶塞。一旦血液吸入CPT,就可将其离心分离PBMC,然后运送(如果需要)到加工实验室。处理实验室从管的上部隔室(凝胶塞上)除去PBMC,洗涤PBMC,并可以使用含DMSO介质冷冻保存,如本方案所述。

背景 外周血单核细胞(PBMC)的分离和低温保存在采用细胞免疫测定的临床研究中是常见的做法。冷冻保存允许样品的批量化,这是方便的并且提高了数据的可比性。冷冻保存还允许将细胞存储在将来未知的未知目的。因为红细胞和粒细胞比冻融融化要脆弱得多,所以PBMC分离是血液细胞冷冻保存的常见先决条件。 PBMC分离最常见的方法是使用高分子量碳水化合物溶液Ficoll-Hypaque进行密度梯度离心。
 含有Ficoll-Hypaque的细胞制备管(CPT)以两种方式简化了密度梯度离心的标准方法:(1)将血液收集到同一管中,然后用于分离PBMC;和(2)管预装有Ficoll-Hypaque,其由凝胶塞分离,使得其不会被血液进入管中的干扰。抽血后,离心管,PBMC和血浆通过凝胶塞与红细胞和粒细胞分离(见图1)。这样可以运送纺丝管,将PBMC保持在与红细胞和粒细胞分离的环境中,这可以提高其生存力和功能。 ...

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