| Detection and Quantification of African Swine Fever Virus in MA-104 Cells
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Author:
Date:
2021-03-20
[Abstract] Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus aethiops) kidney epithelial cells, supports the detection of ASFV from field samples with a sensitivity comparable to that of primary swine macrophages. Collection of swine blood or lungs is time costing, which is often not readily available in most veterinary diagnostic laboratories. MA-104 cells could thus be used as substitute for primary swine macrophages to save significant lead time by avoiding the production of primary swine macrophages. ...
[摘要] [摘要]活的非洲猪瘟病毒(ASFV)的检测在历史上一直依赖于原代猪巨噬细胞(PSM)的使用。PSM不能复制,必须从供体猪中新鲜分离。我们先前发现,MA-104细胞(ATCC#CRL-2378.1)是一种从非洲绿猴(Cercopithecus aethiops )肾上皮细胞分离的商业细胞系,支持从野外样品中检测ASFV,其灵敏度可与ASFV媲美。原发性猪巨噬细胞。Ç的猪的血液或肺ollection是时间成本计算,这往往是在大多数兽医诊断实验室容易获得的。MA-104细胞因此可以用作原代猪巨噬细胞的替代品,通过避免原代猪巨噬细胞的产生来节省大量的准备时间。
[背景]非洲猪瘟病毒(ASFV)的成员,非洲猪瘟病毒科的家庭,导致野猪和家猪具有高度传染性和致命性出血热,即非洲猪瘟(ASF)。成熟的病毒颗粒(病毒粒子)直径为175-215 nm,脂质双层包裹了二十面体衣壳和180-190千碱基对的双链DNA基因组。根据宿主特征和病毒株,该病毒会引起多种症状,包括高度致死性至亚临床性(Tulman等,2009 ...
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| Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs)
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Author:
Date:
2021-02-05
[Abstract] Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for ...
[摘要] [摘要]人类诱导的多能干细胞(iPSC)及其后代具有组织特异性,为再生医学的研究铺平了道路,并在疾病的病理机制阐明和候选药物的发现等领域进行了研究。iPSC集-来源的神经元是特别有价值的,因为它是难以分析神经细胞获自人类的中枢神经系统。对于用iPSC诱导神经元,最常用的方法之一是在形成胚体(EB)之后分离和扩展神经玫瑰花结。然而,该过程费力,效率低下,并且需要进一步纯化细胞。为了克服这些限制,我们已经开发出一种高效神经诱导方法,该方法允许来自于7天内的iPSC神经干/祖细胞(NSCs / NPC的)的产生和功能的成熟神经元的。我们的方法产生一个PAX6 -阳性同质细胞群中,皮质神经干细胞/ NPC的,和t他所得的NSCs / NPC的可冷冻保存,膨胀,并分化在功能性成熟神经元。此外,我们的协议将比其他方法便宜,因为该协议在神经诱导期间需要较少的神经补充。本文还介绍了FM1 - 43成像测定法中,其是用于所述的iPSC衍生的突触前评估中有用的人类神经元。该协议为生成NSC / NPC和神经元提供了一种快速且简化的方法,使研究人员能够建立体外细胞模型来研究脑部疾病的病理学。
[背景]人类iPSC于2007年通过使用四种转录因子(Oct4,Sox2,Klf4和c-Myc)对皮肤成纤维细胞进行重编程而首次建立,并且表现出与胚胎干细胞(ESCs)相似的特征,包括其多能性和自我-更新(Takahashi等,2007; ...
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| Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System
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Author:
Date:
2021-01-20
[Abstract] Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear ...
[摘要] [摘要] 免疫耐受和应答都很大程度上由抗原呈递细胞(APC)表达的主要组织相容性复合物(MHC),T细胞上的T细胞受体(TCR)及其同源抗原之间的相互作用驱动。相互作用障碍导致自身免疫性疾病(例如1型糖尿病)的发病机理。因此,鉴定自身反应性T细胞的抗原表位导致治疗和生物标志物的重要进展。下一代测序方法可从单个T细胞快速鉴定数千种TCR克隆型,因此需要确定已鉴定TCR的同源抗原。该协议描述了T细胞活化的报告系统,其中荧光报告蛋白ZsGreen-1由活化T细胞的核因子(NFAT)信号驱动并通过流式细胞仪读取。记者T细胞也组成性表达额外的一对荧光素tein作为识别物,允许同时多路复用多达8种不同的报告T细胞系,每种表达不同的目标TCR,可通过流式细胞仪区分。一旦制成TCR表达细胞系,仅需一个转导步骤即可将其无限期用于制备新的T细胞系。这种多路复用系统允许筛选TCR-抗原相互作用的数量,否则这些相互作用将是不切实际的,可在多种情况下使用(即,筛选单个抗原或抗原库),并可用于研究任何T细胞-MHC-抗原三分子相互作用。
[背景] T细胞,抗原呈递细胞(APC)及其同源抗原之间的相互作用是自身免疫性疾病(例如1型糖尿病)的主要事件(Michels等,2017; ...
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