Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1.维持巨噬细胞系<ol><li>成长的RAW264.7并在DMEM J774A.1细胞系在5％CO 2的存在下在补充有10％胎牛血清（FBS）中，在37ºC。为了帮助保持无菌，加1％青霉素/链霉素（青霉素/链霉素）的培养基（尽管这不是严格必需的）。 </li><li> 关键的步骤:确保巨噬细胞生长在一个健康的状态，实现高效转染和siRNA诱导的基因敲除。利用通道3和10之间的单元格后新鲜解冻，这些巨噬细胞系具有最佳的转染效率，并保持强劲的转染后的生存在这个阶段;不使用更高的通道数（&gt; 20），作为转染效率和基因敲低既降低很大。 </li><li>对于优化的siRNA转染，细胞板在20％汇合和生长一至两天直到细胞达到不超过80％汇合。杂草丛生的细胞不会转efficiently。 </li></ol> 2.编程96-穿梭系统的转染<ol><li>启动连接到所述梭中的计算机上的96孔穿梭软件。从左上角的文件菜单中新的参数文件 。 </li><li>突出显示将要在显示的96孔板概略转染孔中。绿色方框中会显示该井被选中。 </li><li>选择对应于巨噬细胞系的使用（DS-136对RAW264.7或DS-130对J774A.1），然后选择适用 ，位于diagramed 96孔板下面的程序。请注意，选择一个节目后，在原理图上板的孔将被着色深蓝色。空圆圈表示尚​​未分配的程序并不会感到震惊的井。 </li></ol> 3.准备试剂转染<ol><li>通过混合SF细胞系准备工作Nucleofector™解决方案的96孔Nucleofector™解决方案使用所附的补充管的全部内容。允许该混合试剂的平衡在使用前室温。储存于4ºC过量完整Nucleofector™溶液长达3个月，以备将来使用。 </li><li>预暖的DMEM，RPMI-1640，0.25％胰蛋白酶/ EDTA，和磷酸盐缓冲盐水（PBS），在37ºC之前制备的巨噬细胞用于转染。 </li><li>通过融化冰准备的siRNA转染。为了最小化siRNA的冷冻解冻的数量，以等分试样冷冻新的siRNA首次使用它们。 </li><li>在最初的实验中，使用的siRNA以相对高的剂量为2微米（从20μM的股稀释1:10）。随后滴定的siRNA诱导强有力的基因敲除下到较低的剂量，虽然需要通常较高剂量为有效的基因敲除中的巨噬细胞相比，其他类型的细胞。 注:siRNA是可从许多来源;相当可以获得健壮的基因敲除使用siGenome SMARTPOOLs，这是四个siRNA的靶向每个基因库。作为阴性对照，使用各种非靶向siRNA池或siRNA的。 </li><li>以监测转染效率，转任pmaxGFP对照质粒（在Amaxa公司的试剂盒提供），或荧光标记的siRNA在平行于该实验的siRNA。期望的GFP质粒的30-50％的转染（用显微镜测定或在转染后流式细胞术当天）和荧光标记的siRNA的&gt; 99％的转染（转染和恢复之后通过流式细胞仪立即测定）。 </li></ol> 4.准备的巨噬细胞的转染<ol><li>通过胰蛋白酶消化分离从细胞培养皿贴壁的巨噬细胞。吸出培养基，并用10ml PBS洗细胞两次。除去PBS后，孵育细胞预温的0.25％胰蛋白酶/ EDTA（1毫升/ 10cm平板）1-2分钟，直至细胞开始分离。轻轻晃动酶标板，移动SOLU在孵化期间重刑最大化胰蛋白酶。 </li><li>培养后，通过移液添加9毫升DMEM + FBS的培养基，轻轻混匀，上下，并且收集细胞到无菌管中。 </li><li>算使用血球分离的巨噬细胞的数量。预计每10cm平板大约一千万的巨噬细胞。计算出所需的巨噬细胞的总数;每个孔的96孔的穿梭需要200,000个细胞。记得添加了一些额外的井的价值单元，以便吸取损失。 </li><li>吸取细胞的数目计算为一个离心管中。离心细胞10分钟，于150×g离心室温。不要离心太快，因为这会降低巨噬细胞的健康和转染效率。 </li></ol> 5.用siRNA巨噬细胞的核转<ol><li>使用无菌的96孔圆底板（这同时减少试剂损失便于混合），加入2微升每种siRNA，每孔为互感器ected。 注意:根据siRNA的数目，这可以在巨噬细胞的离心步骤来完成的。 </li><li>从离心细胞吸出培养基。轻轻悬浮细胞Nucleofector™解决方案SF（含补充液），用每200,000细胞沉淀20微升。一旦重悬，立即用细胞最佳转染。 </li><li>传输重悬的细胞进入无菌槽;使用多通道移液管，移20微升的重新悬浮的细胞混合物放入96孔板中含有siRNA的各孔中。移液器向上和向下轻轻混匀。 </li><li>转移20微升细胞/ siRNA的混合物放入96孔Nucleocuvette™板的关键步骤:避免在转移步骤期间产生气泡，因为这会核转期间引起的错误。 </li><li>将所有的转板实验样品后，覆盖96孔板用所提供的盖和版本Ÿ轻轻拍打在坚硬的表面，从样本中删除任何气泡。 </li><li>将96孔板盖子放入96孔Nucleofector班车并选择程序屏幕的右下方"上传并开始"。前核转，按照程序提示保存结果。 注意:在核转过程中，井成功转染将由一个绿色圆圈加号所描绘的96孔原理图在屏幕上。一个红色的圆圈，减号指的错误，很可能是由于气泡或错误吸取试剂的正确数量。 </li></ol> 6.恢复和巨噬细胞的电镀<ol><li>立即核转后，用多道移液器，加入80微升预温（37℃）的RMP1-1640媒体到每个孔中。媒体添加到每孔中的侧面，允许媒体慢慢与转染的细胞，这是非常微妙的，在这个阶段混合。不要添加媒体太快，因为这将大大降低生存能力。 </li><li>将96孔板用转染的样品和RPMI-1640成37ºC培养箱，用5％CO 2下2分钟;这是至关重要的，以使细胞进一步操作之前恢复。 </li><li>从培养箱中取出96孔板中并小心地从每孔中转移到混合物用多道移液器96孔组织培养板中。 </li><li>使用多通道移液管，加入100微升预温（37℃）的DMEM + FBS培养基中的每个孔中。 </li><li>孵育96孔组织培养板用在37ºC培养箱，用5％CO 2下24-36小时的样品（优化siRNA个体的精确定时，尽管该时间段通常可以很好）。 </li></ol> 7. LPS刺激巨噬细胞<ol><li>继24-36小时后核转孵化，准备LPS（或其他的PAMP）的新鲜培养基。准备足够的新鲜培养基对所有井（200微升米EDIA /孔的96孔板加一点点额外的移液）。稀释的LPS至20毫微克/毫升的DMEM + FBS的最终浓度。 </li><li>从96孔板小心吸出培养基，并添加含有内毒素的细胞的新鲜培养基。 </li><li>监控刺激后在不同时间对LPS反应或其它的PAMP。 6小时是足够的，以产生细胞因子如IL-6或TNFα的一个健壮的炎性细胞因子应答。 </li></ol> 8.监控诱导先天免疫反应，基因敲除和活力的巨噬细胞<ol><li>并监控LPS诱导的细胞因子的产生，吸管从细胞上清液中加入96孔储藏板。细胞因子产生可以然后通过ELISA进行测量。 </li><li>一旦除去上清液，用荧光素二乙酸酯监测细胞生存力（当被细胞酯酶活细胞裂解，这种化合物变成荧光）。确定采用t控制细胞活力的siRNA靶向基因他的做法。注:转染过程本身应该有生存力的影响不大，如果执行正确。 <ol><li>添加荧光素二乙酸酯（5毫克/毫升的库存在丙酮）至每个孔中（1:在PBS 100的最终稀释度）。 </li><li>在室温下孵育一至五分钟，然后监测荧光在使用板读数器（460nm处激发，515nm处发射）的细胞。 </li><li>可选:要确保在孔中的荧光均匀，裂解使用缓冲剂如RIPA细胞;这提供了在井均匀的荧光信号，因为有些板读取器被配置为监测井的一小部分;如果细胞不是均匀地镀敷，荧光素二乙酸酯可以得到不准确的读数。 </li></ol></li><li>作为替代，以监测细胞生存力，监视的siRNA诱导的基因使用从贴壁细胞的RNA通过qPCR拦截。 <ol><li>之后将上清液从细胞中除去（ 步骤8.1以上），广告ðRLT缓冲液（其用于裂解和保存的RNA）直接连接到贴壁细胞以裂解它们。 </li><li>使用RNA制备试剂盒，如的RNeasy迷你试剂盒提取RNA，并使用定量PCR用基因特异性引物并监控基因敲除相对于对照siRNA处理。使用引物对Βactin或GAPDH用于标准化。 </li></ol></li><li>作为另一种选择，监察先天免疫应答的其它方面，通过加入FITC标记的大肠杆菌监视巨噬细胞的吞噬能力菌颗粒上直接使用试剂盒的贴壁细胞。按照制造商的协议，它只需几个小时，监视使用读板器或显微镜巨噬细胞吞噬。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。这篇文章是由赞助Lonza公司

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...

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。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

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.

为了证明使用这种方法转染的效率，我们使用流式细胞仪（ 图1）监视的FITC标记的siRNA的摄取。 为了说明我们的用于监视先天免疫应答的方法的实用性，我们转染的靶向已知的先天性免疫调节基因插入的RAW264.7巨噬细胞系的siRNA，刺激了细胞用LPS，然后监测生产的促炎性细胞因子的IL- 6和TNFα。不同的TLR识别不同的PAMP，与从由TLR4 20识别革兰氏阴性细菌的脂?...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

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.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  National Jewish Health and University of Colorado School of Medicine. 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.

视频: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

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