This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

所有的动物实验获得批准的动物护理和使用委员会在得克萨斯大学医疗分支。 来自非感染者和WNV感染的小鼠1.隔离的DC <ol><li>年龄和性别匹配的6-10周龄，野生型C57BL / 6（B6）和MyD88的- / -小鼠。接种腹腔（IP）500空斑形成的WNV NS4B- P38G突变体单元（PFU）。在第3天感染后，安乐死B6和MyD88的- / -小鼠用CO 2。使用这两个群体为对照组的非感染小鼠。使用三只小鼠从每个组。 </li><li>对处死小鼠用70％的乙醇左侧湿毛皮和切去毛发沿着鼠标的左侧，对正面和背面的腿之间中途。剖开体腔。通过使用镊子取出脾脏。将脾脏在培养皿用RPMI。 </li><li>通过根据使用抗CD11c磁珠分离脾DC制造商的说明。 </li></ol> 2.纯化及的OTII转基因小鼠T淋巴细胞标签<ol><li>从天真OTII鼠标收获1脾如上面的步骤1.2描述和同质化的两个幻灯片磨砂两端之间的脾脏。细胞悬液转移到15毫升锥形管中，加入RPMI培养基高达14毫升让该悬浮液静置5分钟，并转移13毫升它以一个新的15毫升锥形管中。 </li><li>通过使用根据制造商的说明一个CD4 + T细胞分离试剂盒分离脾CD4 + T细胞。 </li><li>转移细胞到50ml锥形管中，用PBS洗两次。重悬的细胞在PBS中与0.5％牛血清白蛋白（BSA）以每毫升1×10 6个细胞，并加入CFSE 0.5微摩尔/毫升。孵育在37℃进行10分钟，避光。 </li><li>加入5毫升冷的完全培养基（RPMI-1640，用10％热灭活的胎牛血清，1/100体积抗生素/抗真菌剂，1/100体积L-谷氨酰胺和1 / 1,000（体积）的1000×2-巯基乙醇）。 </li><li>在冰上孵育5分钟。洗涤细胞一次，用PBS，固定0.2×10 6个标记的CD4 + T细胞在2％PFA中并获取关于流式细胞仪。使用该样品，以确定CFSE的基础水平。重新暂停标记的细胞在完全培养基中的其余部分。 </li></ol> 3.共培养OTII T细胞与WNV感染小鼠树突状<ol><li>培养2×10 OTII小鼠5纯化CD4 + T细胞单独或与野生型或MyD88的纯化的DCs - / -小鼠（2×10 4）在一个24孔板有或没有残余的OVA 323-339（1克/毫升）的1毫升每孔完全培养基。 </li><li>孵育细胞在37℃5天。收获细胞，在FACS缓冲液洗两次（PBS中的1％FBS）和0.25ml的2％PFA固定。涡马上。 </li></ol> 体外 4.流式细胞仪分析&lt;/ EM&gt; T细胞活化<ol><li>对于收购，双击流式细胞仪软件图标的流量。对于线性FSC-A 与线性SSC-A的密度图，选择通道0到20万FSC-A和0至20万SSC-A。然后创建活细胞上的栅极（P2;参见图1A）。 </li><li>分析CFSE荧光强度，打开直方图为FL1通道并获取关于门控人口20000事件。上设置的样品的标志物OTII细胞单独培养（ 图1A）。 </li><li>以类似的方式的DC（ 图1C） -采集样品OTII细胞共培养与野生型的DC（ 图1B）或MyD88的- /。 </li></ol> 5.分离的脾细胞为刺激细胞因子测定<ol><li>如STE描述的小鼠与用同样的步骤西尼罗病毒NS4B-P38G突变体-感染B6和MyD88的- /P 1.1。在第30天感染后，再挑战存活小鼠与2000 PFU野生型WNV菌株的ip在天8和21后的主WNV感染或在4天之后继发感染，安乐死的小鼠用CO 2收集脾脏。 注意:我们使用每组2-3只小鼠每个时间点。 </li><li>使脾细胞的单细胞悬浮液如在步骤1.2和步骤2.2，如上所述。计数使用血球和细胞在10ml完全培养基中重新悬浮它们。 </li><li>稀释细胞用完全培养基，以2.5×10 6个细胞/ ml。加入1毫升脾细胞在1.5ml微离心管中。 </li><li>以模拟的CD8 + T细胞，稀释的WNV特异性NS4B和E肽（SSVWNATTA和IALTFLAV）至1mg / ml，在DMSO中。对于CD4 + T细胞的刺激，稀的WNV特异性NS3和E肽（RRWCFDGPRTNTILE和PVGRLVTVNPFVSVA）至1mg / ml，在DMSO中。添加10微升肽至followe细胞ð1微升的布雷菲德菌素一个解决方案。用无细胞治疗肽作为对照。的细胞混合。 </li><li>冲两个孔与18G的针头，在管的盖子。孵育细胞在37℃5小时。 </li></ol> 6.内细胞因子染色<ol><li>转移细胞到新的微量离心管中。旋转细胞5分钟在300-400 XG和倒出上清液。加入1毫升流式细胞仪缓冲，旋转5分钟在300-400 XG和移液用120微升FACS缓冲液重新悬浮细胞。 </li><li>添加2微升的Fc受体阻滞剂孵育细胞，在室温下放置10分钟。加入1 ml FACS缓冲液到每个管中。旋转5分钟，在300 - 400×g的。 </li><li>在300微升FACS再悬浮细胞缓冲剂和它们分成三个管（每管约0.8×10 6个细胞）。如表1所示，保留的各培养条件大鼠IgG-PE染色第一管。对于第二个管中，加入3微升抗CD4的APC与CD4肽处理的细胞，3μ升抗CD8 FITC对CD8肽处理的细胞，或两种抗体对细胞无肽治疗。使用第三管补偿染色。对于每个培养条件，使用细胞染色与APC结合的CD4，FITC结合的CD8或PE-标​​记CD3抗体单独（每管3微升）作为FL1，FL2和FL4渠道补偿控制的三级管。 </li><li>涡细胞短暂。置于冰上20分钟，避光。添加1.4毫升FACS缓冲。旋转5分钟在300-400 XG和倒出上清液。 </li><li>煽动扰乱细胞沉淀（或简称涡）。再暂停250微升固定/通透solutionby吹打细胞沉淀。在室温下孵育20分钟，并避光。 </li><li>添加1.2毫升FACS缓冲。旋转5分钟在300-400 XG和悬浮细胞在300微升FACS缓冲。 注意:在这一点上，细胞可以转移到BL2实验室用于进一步处理或贮存在冰箱中和避光为üp来三天。 </li><li>加入500μlFACS缓冲。旋转5分钟在300-400 XG和悬浮细胞在500微升1个通透/洗涤缓冲液中。旋转5分钟在300-400×g的。 </li><li>重新暂停细胞在100微升1个烫发/洗净，加3.5微升抗IFNγ-PE先前与抗体的CD4和/或CD8 + T细胞标记步骤6.3和3.5微升大鼠IgG-PE预留管加管对于IgG对照在冰上25分钟，避光。将1.4毫升1×通透/洗涤缓冲液洗涤细胞。旋转5分钟在300-400×g的。通过加入1.4毫升1X通透/洗涤缓冲液重复洗涤步骤。 </li><li>旋转5分钟在300-400 XG，倒出上清液。将1.4毫升的FACS缓冲液，并重新悬浮于400μlFACS缓冲液进行最后采集洗涤细胞。 </li></ol>内细胞因子染色7.流式细胞仪分析<ol><li>使用相同的采集的设置步骤4.1。 CREA德活细胞上的栅极（P2，参见图2A）。调整使用补偿管的每个组的电压。 </li><li>打开两个新的散点图，一个用于显示收集FL1 VS数据。 FL2（CD8 VS IFN-γ），另一种是显示收集FL4 VS的数据。 FL2（CD4 VS IFN-γ）。获得50,000个事件对于各样品的门控群体。 </li></ol>

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作者什么都没有透露。

WNV是BL3病原体。由于安全法规，免疫学检测与WNV感染的样本通常是由设备的可用性BL3设施或者更加漫长和繁琐的进行限制。在最近的研究中，我们使用了两种流式细胞仪为基础的方法西尼罗病毒感染5时，研究细胞介导的免疫反应。在这两个试验中，西尼罗病毒感染的细胞用直接或用含有4％PFA固定/透化溶液1-2％的PFA。众所周知，病毒感染的细胞的1％PFA中固定可有效降低到低于检测限1...

西尼罗河病毒（WNV），神经营养，加黄病毒检测到，是一个新兴的公共健康威胁。目前，还没有疫苗已被批准供人类使用1。减毒WNV菌株，其具有P38G取代的非结构（NS）蛋白4B，被称为诱导小鼠没有杀伤力但较高先天细胞因子和T细胞应答的小鼠比野生型WNV NY99菌株2。免疫小鼠的NS4B-P38G突变均免受致命野生型WNV次要挑战。这表明，NS4B-P38G突变体具有合适特征的理想的候选疫苗。通过该NS4B-P38G突变导致的高保护性免疫机制尚不清楚呢。 toll样受体（TLR），其识别病原体相关分子模式，发挥在先天免疫病毒感染的启动中起重要作用。核心TLR信号通路利用骨髓分化初级反应GENE 88（MyD88的）作为主适配器3,4。在最近的一项研究中，MyD88信号被证明在小鼠5 WNV NS4B- P38G突变感染在细胞介导的免疫发展具有重要作用。树突状细胞（DC）是最重要的抗原呈递细胞表现出病毒感染6,7中以引发初级T细胞反应的独特能力之一。 CD4 +和CD8 + T-细胞既有助于长期的保护性免疫，并且下面的野生型WNV感染8,9-重要寄宿存活。两种免疫测定法在本研究中使用，以评估这些细胞在NS4B-P38G突变感染的小鼠的功能。 首先， 在体外 T细胞吸测定用于比较的DC西尼罗病毒感染的野生型和MyD88的的抗原呈递能力- / -小鼠。为了提高检测，天真CD4 &lt;灵敏度SUP&gt; + T细胞从OTII的转基因小鼠，其表达Vα2/Vβ5TCR特异性鸡卵白蛋白（OVA）肽323分离- 339.议会西尼罗病毒感染小鼠进行纯化，并共培养以羧基琥珀酰亚胺复活节（CFSE ）标记的CD4 + T细胞在OVA肽存在下进行。经过5天共培养，收获细胞并固定用多聚甲醛（PFA）和通过流式细胞术分析。增殖测定法已被传统上进行的，通过将5-溴-2&#39;-脱氧尿苷（BrdU）标记或氚化胸腺嘧啶脱氧核苷（3 HTdr）10掺入。然而，这些测定是要么放射性和/或需要在生物安全等级（BL）的3设施，其中，西尼罗病毒的研究进行的专用设备。的淋巴细胞增殖的活体染料CFSE标记已经变得更加普遍的免疫学测定法中使用的荧光强度的串行减半的流式细胞仪分析的染料更稳定地并均匀地掺入到细胞中，用流式细胞容易检测仪，并且非放射性11。该测定法还具有以评估细胞分裂的数目的能力。使用这种测定法中的WNV研究的一个主要优点是，感染的细胞以1-2％的PFA的固定可以灭活西尼罗病毒12，这将使样品采集在一个BL2实验室流式细胞仪。 接着，经修饰的细胞内细胞因子染色（ICS）的步骤，用于研究的MyD88的信令中的WNV特异性T细胞应答调节NS4B-P38G突变感染的小鼠中的作用。在该测定中，脾细胞从感染小鼠分离的处理在体外用WNV特异性肽。布雷菲德菌素A加入到保持在细胞内的细胞因子。后5小时温育后，收获细胞，洗涤并染色T细胞亚群。然后将细胞固定在PFA，透化染色对干扰素（IFN）-γ和通过流式细胞术分析。与其他术为基础的流测定，一旦细胞被用固定和含有PFA透化缓冲液处理过的，被感染的样品可以被转移到一个BL2实验室用于进一步处理和分析。在一些发表的研究中，我们使用的ICS测量在WNV感染的小鼠13,14的T细胞的效应子功能。虽然它的确定无疑的，此测定中的一个主要缺点是，该过程是非常冗长并且可能是更多的时间内BL3设施执行时费时。这里，微离心管系的ICS方法被证明是更可行的，更容易进行，以及更短的时间时内BL3实验室进行耗时。

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI 1640</td>
			<td style="width:153px;">Invitrogen</td>
			<td align="right" style="width:119px;">11875</td>
			<td>warm up at 37C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD11c magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD4 magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-248</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td style="width:153px;">Invitrogen</td>
			<td>C34554</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">OVA residue 323-339</td>
			<td>Genscript Corporation</td>
			<td>RP10610</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
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			<td height="39" style="height:39px;width:216px;">Peptides</td>
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			<td>PC0AD-D</td>
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		<tr height="58">
			<td height="58" style="height:58px;">Brefeldin A solution</td>
			<td style="width:153px;">BD Bioscience</td>
			<td align="right">555029</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Mouse Fc Blocker</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>14-0161-85</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">APC-conjugated CD4&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>17-0041-81</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">FITC-conjugated CD8&#160;</td>
			<td style="width:153px;">e- bioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Fixation/Permeabilization Solution</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right" style="width:119px;">554722</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Permeabilization/wash buffer</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right">554723</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">anti-IFNg-PE&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>12-7311-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Accuri flow cytometer</td>
			<td style="width:153px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

减毒西尼罗河病毒（WNV），非结构（NS）的4B-P38G突变，诱发较高先天细胞因子和T细胞应答比野生型WNV小鼠。近日，髓样分化因子88（MyD88的）信号被证明在WNV NS4B-P38G突变感染是初始T细胞吸和记忆性T细胞的发育很重要。在这项研究中，2流式细胞术为基础的方法- 在体外的T细胞引发测定和胞内细胞因子染色（ICS） -被用于评估树突细胞（DC）和T细胞的功能。标记OTII转基因小鼠的CD4 + T细胞-在T细胞吸试验中，细胞增殖通过流式细胞仪从两组小鼠羧基琥珀酰亚胺酯（CFSE）的下列DC的共培养物进行分析。这种方法提供了一种准确测定增殖的CD4 + T细胞的百分比与显著改进比传统的整体灵敏度人检测放射性试剂。微量离心管系统被用来在ICS协议的细胞培养和细胞因子染色程序。相较于传统的组织培养板为基础的系统，这个修改过程是比较容易在生物安全水平（BL）3设施进行。此外，WNV-感染的细胞与多聚甲醛在两种测定中，这使BL3设施以外进一步分析处理。总之，这些体外免疫学测定法可以用来有效地评估WNV感染期间细胞介导的免疫反应。

在T细胞吸试验中，CFSE标记的CD4 + T细胞是从NS4B-P38G突变体感染的野生型和MyD88的纯化的DC培养- / -小鼠中OVA肽的存在或不存在。标记的T细胞单独培养有或无OVA 5天用作阴性对照。 如图1A所示 ，总T细胞被门控为荧光强度对FL1通道分析。标记成立基于所述新鲜标记的CD4 + T细胞在第0天以确定细胞增殖速度，而不DC的共培养物。有这种培养条件下增殖率（1.6％）的低?...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

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 ...

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Research

JoVE Journal

Immunology and Infection

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

8.1K 次观看.  The University of Texas Medical Branch. Two flow cytometry-based methods – 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.

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

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. ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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.

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...