| In vitro Measurement of Membrane Attack Complex in RPE Cells
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Author:
Date:
2021-02-20
[Abstract] Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the outer blood retinal barrier. RPE cells are highly polarized with apical microvilli and basolateral contact with Bruch’s membrane. In order to obtain biologically relevant polarized RPE cultures in vitro, RPE cells are seeded onto the apical side of a transwell filter and cultured for 4 weeks in low serum media. MAC formation on RPE cells has been reported to be sub-lytic. MAC formation can be achieved in vitro by introduction of normal human ...
[摘要] [摘要]补体系统的启动导致形成称为膜攻击复合物（MAC，C5b-C9）的多蛋白孔。MAC孔积聚在细胞表面，可导致细胞裂解。视网膜色素上皮细胞（RPE）是位于眼那种形式的后轮询色素上皮细胞的单个单层š外血视网膜屏障。RPE细胞高度极化，顶端微绒毛和与Bruch膜的基底外侧接触。为了在体外获得生物学上相关的极化RPE培养物，将RPE细胞接种到Transwell滤膜的顶端，并在低血清培养基中培养4周。MAC形成Ò Ñ据报道，RPE细胞是亚裂解的。通过在血清饥饿24小时后向培养基中引入正常人血清（NHS），可以在体外实现MAC的形成。NHS包含启动补体激活和MAC形成所需的所有血清补体蛋白。我们结合了体外RPE极化和补体激活，以利用共聚焦显微镜在体外可视化MAC形成，从而实现了高分辨率MAC成像。
[背景]补体系统是一种进化保守的先天免疫途径。补体激活存在三种主要的独立但重叠的途径，它们在C3转化酶，经典途径，凝集素途径和替代途径中收敛。在经典途径中，免疫复合物（抗原-抗体复合物）通过C1q亚成分结合C1，然后C1s蛋白酶亚基裂解补体因子C4和C2。这些片段（C4bC2b）形成酶复合物“ ...
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| In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
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Author:
Date:
2020-09-20
[Abstract] Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol ...
[摘要] [摘要 ] 诱导性多能干细胞衍生的心血管祖细胞（iPSC-CVPCs）为检查心脏发育和疾病病因的分子基础提供了前所未有的平台，但在再生医学和药物基因组学的未来中也具有重要作用。像iPSCORE这样的生物库（Stacey 等，2013 ；Panopoulos 等，2017）， 其中包含由数百个遗传和种族不同的个体产生的iPSC，是进行这些研究的宝贵资源。在这里，我们为小分子大规模衍生人iPSC-CVPCs和代谢选择纯化提供了一种优化，具有成本效益和高度标准化的方案。我们已经成功地应用了该协议，从154种不同的iPSCORE iPSC品系中获得了iPSC-CVPC，从而获得了大量的高纯度心脏细胞。一个重要的我们的协议的组成部分是Ç ELL Ç onfluency 估计S（ccEstimate ），用于估计当iPSC集单层将达到80％汇合，这是用于发起的iPSC-CVPC推导最佳的时间的自动方法，并且使得协议为易于在具有不同增长率的iPSC系列中使用。此外，我们发现跨iPSC-CVPC的细胞异质性是由于两种截然不同的心脏细胞类型（心肌细胞（CMs）和心外膜衍生细胞（EPDCs））的比例不同导致的，这两种细胞在心脏再生中均具有关键作用。该协议消除了iPSC线到线优化的需要，并且可以轻松地进行调整和扩展，以进行高通量研究或生成大量适用于再生医学应用的细胞。
[背景 ] ...
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| Identification of Socially-activated Neurons
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Author:
Date:
2020-09-05
[Abstract] Determining the neuronal circuitry responsible for specific behaviors is a major focus in the field of neurobiology. Activity-dependent immediate early genes (IEGs), transcribed and translated shortly after neurons discharge action potentials, have been used extensively to either identify or gain genetic access to neurons and brain regions involved in such behaviors. By using immunohistochemistry for the protein product of the IEG c-Fos combined with retrograde labeling of specific neuronal populations, precise experimental timing, and identical data acquisition and processing, we present a method to quantitatively identify specific neuronal subpopulations that were active during social encounters. We have previously used this method to show a stronger recruitment of ventral hippocampal ...
[摘要] [摘要] 确定负责特定行为的神经元回路是神经生物学领域的主要重点。在神经元释放动作电位后不久就转录和翻译的依赖活动的立即早期基因（IEG）已被广泛用于识别或获得涉及此类行为的神经元和大脑区域的遗传途径。通过对IEG c- Fos 的蛋白质产物使用免疫组织化学，结合特定神经元群体的逆向标记，精确的实验时间，相同的数据采集和处理 ng，我们提出了一种方法，可以定量地识别在社交活动中活跃的特定神经元亚群。与社交活动后投射到下丘脑外侧的神经海马神经元相比，我们以前曾使用这种方法显示出投射到内侧前额叶皮层的腹侧海马神经元更强的募集。在优化用于逆行示踪剂注射的手术后，该方法将有助于识别和定位参与许多不同行为的神经元群体。
[背景] 神经生物学领域的基本目标是确定大脑如何处理感觉输入并产生行为输出命令。在过去的二十年中，某些大脑区域已成为控制特定行为的典范实例，例如海马体用于位置记忆（Bird和Burgess，2008年）。最近的工作开始以可以推广到多种行为的方式来解析这些区域的特定功能（Aronov 等人，2017），并确定在由远程神经元连接的给定行为中多个大脑区域的作用投影（ ...
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