Name: optimized protocol for efficient transfection of dendritic cells without cell maturation
Uploaded: 2011-07-08T13:00:00
Description: Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

该项目是由美国国立卫生研究院NIAID的合同号HHSN2662000500021C支持。我们感谢他的技术援助名臣。

1。计划Amaxa 96穿梭Nucleofector <ol><li>打开一个新的参数文件。 </li><li>选择您将使用标准的转拖动光标在96孔板图，井的数量。使用至少3口井，池，每个实验样品。 </li><li>输入程序代码：在第一部分选择“FF”，并在第2部分选择&#39;168&#39;从下拉式菜单</li><li>从解决方案中，选择“单核细胞，人类” </li><li>在控制选项，选择“标准”。 </li><li>点击应用。 </li><li>包括无染控制，从图中选择进一步根?...

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幼稚的初级树突状细胞的高效转染重要的是，在这关键的细胞介导的​​先天适应性免疫过渡的细胞炎症通路的高通量分析和逆向工程。然而，大多数研究人员发现，这些细胞是难以转染效率和转染过程诱导细胞成熟时，使用标准的转染技术。我们研究了克服这些限制是否可以由高吞吐量协议优化使用，同时，独立96以及商业核转染系统（龙沙）。 利用荧光激活细胞分选（FAC...

<table border="1"><tbody><tr><th>设备/试剂</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td> Amaxa Nucleofector 96以及班车</td><td>龙沙</td><td> 108S0109 </td><td>序列号</td></tr><tr><td> Amaxa人类单核细胞96 Nucleofector套件</td><td>龙沙</td><td> VHPA - 2007 </td><td>包含96 Nucleofector解决方案的人单核细胞，96的补充和Nucleocuvettes和板</td></tr><tr><td> RIG -我的siRNA </td><td> Dharmacon </td><td> L - 012511 - 00 </td><td></td></tr><tr><td> GLO的siRNA </td><td> Dharmacon </td><td> D - 001600 - 01 - 20 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 11875 </td><td>辅以10％的FCS，2毫米L -谷氨酰胺，100 U / ml青霉素和100微克/ ml链霉素，使直流生长介质</td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> Invitrogen公司</td><td> 25030081 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15070063 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> 3070.03 </td><td></td></tr><tr><td>树突状细胞</td><td>纽约血液中心</td><td></td><td>区议会是从Buffy大衣使用的标准程序纯化</td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

树突状细胞可以被认为是其中发挥关键作用，在其发起和响应感染 1的免疫系统的哨兵。天真区议会致病抗原的检测是通过模式识别受体（PRRS）是能够识别特定病原体相关分子模式（PAMPS）的保守结构。区议会PAMPS检测触发细胞内的信号级联，导致他们的活化和转化为成熟DC。这个过程通常是通过对1型干扰素的生产特点以及与其他炎性细胞因子，如MHCII和CD86和迁移的淋巴结，与T细胞相互作用启动适应性免疫反应的成熟DC的细胞表面标志的上调， 3。因此，区议会的联系先天免疫和适应性免疫系统。 解剖的基本直流响应，以各种病原体的分子网络的能力是至关重要的，以更好地了解了这些信号通路的调控和诱导基因。它也应当有助于促进DC为基础的疫苗针对传染病和肿瘤的发展。然而，此行的研究已经严重阻碍，难以转染的主要区议会 4 。 病毒转导的方法，如慢病毒系统，通常使用的，但携带很多限制，如复杂性和有害生物风险（ 相关费用）5,6,7,8。此外，该病毒基因产物的交付增加转区议会 9,10,11,12的免疫原性。电穿孔技术已用于13,14,15结果好坏参半，但我们是第一个报告使用一种高通量的转染协议和确凿证明其效用。 在这份报告中，我们总结了一个优化的商业协议，高通量转染人类的主要区议会，与有限的细胞毒性和一个 DC成熟16的情况下。转染效率（GFP质粒）和细胞活力分别超过50％和70％。流式细胞仪分析建立的成熟标志物CD86和MHCII转染细胞中表达增加的情况下，而定量RT - PCR结果显示没有IFNβ上调。使用此电协议，我们提供证据议会成功转染siRNA和有效的敲有针对性的基因RIG - I，16,17一个关键的病毒识别受体的mRNA和蛋白水平，。

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

我们目前我们优化的高通量nucleofection协议，要么质粒DNA或不会造成细胞的成熟的siRNA转染初级人类单核细胞衍生的树突状细胞的有效方法。我们进一步提供证据证明为成功的siRNA沉默靶基因的mRNA和蛋白水平的RIG - I。

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Research

JoVE Journal

Immunology and Infection

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科研

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人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

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从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...