| An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation
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Author:
Date:
2021-02-20
[Abstract] The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with ...
[摘要] [摘要]中性粒细胞胞外陷阱(NETs)的形成被认为在感染和传播无菌炎症中起关键作用。组蛋白瓜氨酸化是NETs形成中必不可少的早期步骤,可在形成标志性的细胞外DNA支架链之前检测到。除了经典的显微镜方法外,还在研究和临床目的开发用于NETs及其检测的新技术。NET的经典显微镜研究具有主观性,低通量和半定量性,并且捕获早期步骤的能力有限。我们开发了这种新颖的成像流式细胞仪 (IFC)方法,专门识别和量化组蛋白H4的瓜氨酸化作为NETs标记及其与核和细胞水平其他改变的关系。这些包括核解聚和超缩合,多分叶状核与1-叶核和细胞膜损伤。在形成特定的细胞外DNA支架链之前,可以在用NETs诱导剂进行不同的治疗期后对NETs标记物进行定量。由于这些高通量基于图像的细胞分析功能可以通过严格的统计来执行,因此该协议适用于实验和临床应用以及作为生物标志物的NETosis的临床评估。
[背景]活化的中性粒细胞迅速募集到感染和受伤的地方;它们有助于宿主抵抗病原体和炎症。大约在二十年前,首次观察到对病原体的反应是,一部分中性粒细胞会经历细胞凋亡或坏死以外的不同类型的细胞死亡。这涉及染色质的早期缩合,核区室和颗粒区室的共定位,最后将这种混合物释放到环境中。锚定核成分(例如组蛋白)或细胞成分的细胞外DNA支架链,其中一些具有很强的抗病原特性,例如弹性蛋白酶或髓过氧化物酶(MPO),被称为“嗜中性细胞外陷阱”或NETs,并具有有效的抗菌活性(Brinkmann等人,2004)。最近的证据表明,母语也非感染性疾病,包括癌症,系统性关联红斑狼疮,镰状细胞病,动脉粥样硬化和血栓形成(Jorch和Kubes,2017年)。尚不清楚NETs或其任何成分(无细胞DNA或组蛋白)是否作为驱动因素导致了这些疾病的病理,或者它们是否是免疫反应失衡的副产物。NETs在某些疾病中也可能充当生物标志物,并提供有关治疗方案功效的信息(Barnado等,2016)。NETs相关成分(例如弹性蛋白酶或MPO)存在于自身免疫性疾病患者的血浆中,也存在于感染中,这使这些分子作为生物标记物的特异性变得复杂。因此,迫切需要用于NETs表征的新方法,这些方法应在研究之间提供一致的标准,并且对于机械和临床应用均可靠。 ...
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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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| Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
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Author:
Date:
2020-06-20
[Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be ...
[摘要] [摘要] 丝氨酸Palmitoyltranferase (SPT)是吡哆醛5 ' 磷酸(PLP)依赖酶催化第一和限速步骤中从头合成鞘脂。SPT活动是Homeostatically调控响应水平的提高鞘脂。这SPT的稳态调节是通过小ER膜蛋白介导称为ORMDLs。在这里,我们描述了一种方法用放射性标记的L-丝氨酸以测定SPT活性的SPT活性。测定的ORMDL依赖性抑制脂质从由规定的程序被开发Hornemann 实验室。 SPT的活性也可以使用氘化的L-丝氨酸进行测定,但需要进行质谱分析,这会耗费金钱,时间和仪器。可以在细胞和无细胞系统中研究ORMDL依赖性脂质对SPT活性的抑制作用。在这里,我们提供了详细的协议来测量存在短链(C8-神经酰胺)或长链神经酰胺(C24-神经酰胺)时SPT活性。该协议的最大优势之一我们通过在HeLa细胞膜中提供外源鞘氨醇和24:1酰基辅酶A通过内源性神经酰胺合酶生成长链神经酰胺来实现这一目标。需要精密的仪器。
[背景 ] 丝氨酸palmit oyltranferase (SPT)是一种多亚基酶是在真核生物和原核生物一些广泛表达(花田等人,1997; Ikushiro 。等人,2001; Hornemann 等人,2007).The ...
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