利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞
Summary
In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.
Abstract
Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.
Introduction
在这个协议中，我们描述了有效的方法，以抑制使用的siRNA的基因在小鼠巨噬细胞系和监视这些处理上的先天免疫反应的影响。这些程序在96孔格式进行，允许RNAi筛选在中等或高通量的方式。
在对感染的反应，人类装入立即先天免疫反应和较慢的但更具体的适应性免疫反应。这种快速先天免疫反应涉及吞噬先天免疫细胞，包括巨噬细胞1的募集和活化。经典的活化巨噬细胞参与了急性炎症反应，产生抗微生物蛋白和化合物，细胞因子和趋化因子，并呈递抗原2,3。另外活化的巨噬细胞在调节机体免疫力，保持宽容，组织修复的作用，并且伤口愈合4-8。由于他们的职能范围广泛的巨噬细胞可起到多种疾病，包括动脉粥样硬化，关节炎，癌症和9的作用。因此，巨噬细胞的研究一直是研究的重要领域中的各种疾病领域的一段时间。
尽管其在先天免疫应答的重要性，巨噬细胞可以是具有挑战性的细胞来工作。特别是，它难以得到利用巨噬细胞中的脂质试剂有效转染无关联的毒性10,11。此外，即使当的siRNA被有效地传递到巨噬细胞中RNAi诱导的基因经常拦截的鲁棒性可以相当温和，可以从基因而异基因。
为了克服这些技术挑战，我们有两种小鼠巨噬细胞系RAW264.7 17和18 J774A.1优化转染和技术击倒12-16。此方法使用Amaxa公司Nucleofector 96孔班车系统转染;该系统使用专门的试剂和电穿孔的组合转染细胞在96孔格式19。转染后，我们描述的方法来最大化细胞恢复和生存能力，并最大限度地随后的siRNA诱导的基因敲除。为了说明这种方法的实用性，我们描述了一种协议，用于siRNA递送到这些巨噬细胞系，刺激这些细胞用脂多糖（LPS），并监视在生产几种促炎性细胞因子的水平的先天免疫应答。我们提供一种其中我们针对Toll样受体（TLR）家族，其成员感测LPS和其他相关的病原体分子模式（PAMP），以调节先天免疫的样本数据。
Protocol
Representative Results
Discussion
众多研究已经发表，其中各个基因已在小鼠巨噬细胞被设为目标的siRNA。而脂质介导的转染已用于siRNA递送给个体基础上的巨噬细胞的细胞系，这些方法存在的生存能力，有限的基因敲除，以及变异性的潜在影响，从基因到基因。为了开发更强有力的测定法，可以用于靶向基因在中等或高通量的方式，我们的优化使用Amaxa公司nucleofector 96-穿梭系统，它显示出在基因敲除更多的一致性和生存能力有限…
Declarações
The authors have nothing to disclose.
Acknowledgements
Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.
Materials
| Amaxa nucleofector 96-well shuttle system | Lonza | AAM-1001S | |
| Amaxa SF cell line 96-well nucleofector kit | Lonza | V4SC-2096 | |
| RAW264.7 mouse macrophage cell line | ATCC | TIB-71 | |
| J774A.1 mouse macrophage cell line | ATCC | TIB-67 | |
| siGenome smartpool siRNA | Dharmacon | varies depending on gene | |
| Non-targeting control siRNA pool | Dharmacon | D-001206-13-20 | |
| Block-iT fluorescent oligo for electrooration | Invitrogen | 13750062 | |
| Ultrapure E. coli O111:B4 LPS | List Biological Laboratories | 421 | |
| DMEM, high glucose | Invitrogen | 11995-065 | |
| RPM1-1640 | Invitrogen | 11875-093 | |
| Penicillin-Streptomycin Solution (Pen/Strep) | Fisher | SV30010 | |
| 0.25% Trypsin-EDTA | Invitrogen | 25200072 | |
| 96 well tissue culture plates | Fisher | 07-200-89 | |
| 96 well round bottom sterile plates (not coated) | Fisher | 07-200-745 | |
| Mouse IL-6 Duoset ELISA kit | R&D Biosystems | DY406 | |
| Mouse TNFa Duoset ELISA kit | R&D Biosystems | DY410 | |
| Fluorescein diacetate | Sigma-Aldrich | F7378 | |
| RLT Bufer | Qiagen | 79216 | |
| Rneasy mini kit | Qiagen | 74134 | |
| Vybrant Phagocytosis Assay Kit | Invitrogen | V-6694 |
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