作者要感谢奥利维尔曼彻斯，博士，达沃尔Frleta博士和梅根奥布莱恩博士的支持和反馈。这项研究是由过敏和传染病国家研究所的支持，并完成了与美国国立卫生研究院授予露丝属Kirschstein国家研究服务奖个人博士前奖学金（F31），以促进多样性与健康有关的研究（AI089030）和RO1（AI081848资金）。

操守准则：研究样本获得并存储与捐助方的同意研究。所有的样品应该被编码或在使用前匿名。此协议遵循我们的机构审查委员会的指导方针。 1，分化人外周血单核细胞的成单核细胞来源的树突状细胞。 注意：人的血沉棕黄层充当人外周血细胞（PBMC）的源极和来自纽约血液中心（纽约，NY）获得。献血者是健康的志愿者。 5日程序与人外周血单核细胞（PBMC）的电镀开始到组织培养瓶35,36。从公布的协议显着的差异有以下几种： <ol><li>使用225厘米2非致热性聚苯乙烯组织培养瓶用滤帽共每瓶2×10 8外周血单个核细胞，以坚持，而不是10厘米的聚苯乙烯组织培养板秒（58厘米2）（步骤1）。 </li><li>用5％合并的人血清（PHS; 30毫升）中制备介质在500毫升的RPMI-1640 + L-谷氨酰胺，将5ml的1M HEPES缓冲液，并1.4毫升50毫克/毫升庆大霉素（5％PHS介质），然后通过过滤一个20微米的过滤器。加总为50毫升5％小灵通媒体每225厘米2组织培养瓶。 </li><li>用25毫升新鲜的RPMI-1640洗涤粘附细胞3次。每次洗涤过程中剧烈振荡5秒，吸非贴壁细胞（步骤4）。 </li><li>添加190微升400单位/微升的IL-4和380微升100 IU /μl的烧瓶中（步骤3）的GM-CSF在第0天，2，和4。第0天定义为天的PBMC进行初始镀步骤1（步骤8）。 </li><li>收获天5 moDCs在1×10 6个细胞/ ml（步骤15）的浓度。 </li><li>立即在他们休息的状态下使用moDCs。步骤17-22永远不会执行。注：样品应为获得最佳效果，尽快收集后进行处理。 </li><li>分装moDCs在浓度2×10 5个细胞化/孔（200微升1×10 6个细胞/ ml /孔）在96孔圆底平板（免疫印迹，ELISA，FACS），或者添加到聚-L-赖氨酸处理过的chamberslide（显微镜）进行实验。注：至少有4个条件：完全无刺激（阴性对照），吸只，其次是激活只有激活和预充。条件可以扩展，如果需要，包括为R848和稀释剂对照尼日利亚菌素。如果下游分析是ICS的，重复是必要的同型对照。 </li></ol> 2，灌注炎性 - 信号1 <ol><li>根据制造商的说明重组的冻干R848在DMSO中。稀释股票的工作在室温下用RPMI-1640。 </li><li>加R848以最终浓度10μM至适当的孔中18小时。和地点的细胞在培养箱中在37℃，5％CO 2。 </li></ol> 3激活NLRP3炎性体 - 信号2 <ol><li>根据制造商的说明重组的冻干尼日利亚菌素乙醇。添加到适当的条件之前，稀释作业储备，在室温下用RPMI-1640。 </li><li>添加尼日利亚菌素以20μM的终浓度，并返回到培养箱中在37℃和5％CO 2的6小时。吸之间，并在激活过程中无洗涤步骤进行。注：IL-1β的分泌增加了钙离子载体的存在下，布雷菲德菌素A，莫能菌素，二硝基苯酚，或羰基氰化氯苯37,38。因此，除了这些分泌抑制剂，不推荐在分析促发炎性或活动，因为它会改变IL-1β的分泌。 </li></ol> 4，IL-1ß样品采集<ol><li>与细胞和上清液于974×g离心3分钟离心板。 </li><li>而不会干扰细胞沉淀，吸出上清液，并转移到为eparate一轮以从上清液测定细胞因子的分泌见底板。注：上清液可暂时储存在-20℃或-80℃下长期贮存。 </li><li>用200μl的1×PBS中于974×g离心3分钟，洗涤细胞小球3次，以从蜂窝样品除去任何外IL-1β。注意：当洗涤从96孔板中的细胞沉淀，通过迅速板倒置取出洗净入收集箱，以便不干扰样品。 </li></ol> 5，测量IL-1ß从细胞和上清液样品<ol><li>细胞内细胞因子染色（ICS）：该ICS协议进行说明39，40。注意：不要悬细胞，如果下游分析是显微镜。所有洗涤和愿望应不干扰细胞层/丸粒（取决于下游测定）来进行。 <ol><li>加入100μl的5％小灵通介质插入相应的孔中。加合适的体积（约1μl/2x105个细胞，或1微升/孔）的荧光标记的α-CD11c的和α-CD14（表型标志物;克隆B-LY6和MφP9，分别）或同种型对照对细胞进行10分钟，在RT在黑暗中。 </li><li>用1×PBS进行3分钟洗三次，在974×g离心。 </li><li>通过加入100μl的4％PFA中于室温在黑暗中20分钟，固定细胞。 </li><li>暂停点：用100微升的1×PBS以974×g离心3分钟，储存于4°CO / N在1x PBS洗。 </li><li>加入100微升的透化缓冲液对细胞进行30分钟。注意：透化缓冲液组成的1×PBS中与0.3％的Triton X-100和1％牛血清白蛋白（BSA）为透化。请勿打扰贴壁细胞时，下游的测定是显微镜。 </li><li>添加α-IL-1β-FITC的适当体积（约62纳克antibody/2x10 5个细胞，约2.5或100μl/孔）（克隆8516）或同种型对照2小时在恒温箱中在37℃下</li><li>用200μl的透化缓冲液在974×g下在黑暗中3分钟，洗涤细胞3次。 </li><li>可选：染色用DAPI，加封固，并轻轻放置盖玻片的载玻片上。让封固治愈O / N捕获显微图像前。 </li><li>收购流式细胞仪或显微镜的数据。注：同型每个条件比较时用流式细胞仪或显微镜分析适当的染色。 <ol><li>场向电流：获取一个样本的时间。分析的MoDC人口亲IL-1β染色前细胞-在活细胞中，随后选通上的CD11c + CD14设置FSC / SSC门。 </li><li>显微镜：用染色阳性样品设定曝光时间 - R848处理。用DAPI +亲IL-1β+和DAPI +亲IL-1β -确定亲IL-1β+表达moDCs百分比。 </li></ol></li></ol></li><li> SDS-页：免疫印迹法进行检测亲IL-1β。注意：下面介绍的技术标准相结合免疫印迹法，梯度4-20％聚丙烯酰胺凝胶，和荧光或化学发光检测41，42。 <ol><li>裂解细胞中直接加入10μl变性裂解缓冲液（5μl的β-mercaptoethanol/950微升Laemmli样品缓冲液，然后用细胞裂解缓冲液稀释1:1）。裂解物转移至1.5ml离心管中。 </li><li>基于干板10分钟，在100℃下热裂解液</li><li>旋裂解物在20,800×g离心对minicentrifuge 1分钟，以浓缩液体积。暂停点：样品可以在-20℃下冷冻于短期存储。 </li><li>加载的总体积到聚丙烯酰胺凝胶上。运行140伏至约1小时，或在凝胶盒直至染料前沿跑出凝胶的底部。 </li><li>从聚丙烯酰胺凝胶转移蛋白到PVDF的Immobilon-FL membrANE为90分钟，在100 V块，用5％BSA的膜在Tris缓冲盐水/ 0.1％吐温20（TBS-T）1小时。注：PVDF的Immobilon-FL是最适合用荧光检测使用。用不同的组合物的膜，被推荐用于其它检测技术，以增加信号背景比。 </li><li>淡化第一和第二抗体在10毫升5％的BSA / TBS-T的。在4°CO / N对一抗孵育一阵晃动和第二抗体在室温下1小时，同时晃动。 </li><li>用TBS-T洗膜3次，每次5分钟，而抗和二抗孵育，再二次孵化与成像之间晃动。注：可以使用荧光或化学发光检测被检测波段。按照制造商的检测说明。 </li></ol></li><li> ELISA法：被冷冻储存样品需要分析前平衡至室温。降速样品在离心机巩固上清冷凝通报BULLETIN。按照制造商的指示，对IL-1β的测量。注：在本协议中IL-1β是专门测量;然而，TNFα，IL-6，和免疫调节细胞因子IL-10的同时测量确保适当的条件进行底涂。 </li></ol>

该作者有没有利益冲突披露。

炎性细胞因子是一体的操纵先天和适应性免疫反应对抗病毒感染。分泌IL-1β已经证明流感病毒感染3，43，44中增加。由这些细胞因子响应于识别病毒在人树突状细胞加工的精确机制尚不完全清楚。骨髓DC分离试剂盒是昂贵和费时的。分离试剂盒和足总杯的排序可能会无意间应力或激活细胞。此外，还有经常分离的细胞用于实验的量不足。 在体外培养 45，46人原髓细胞DC的还好...

先天免疫系统的活化是必需的感染，疾病，和疫苗接种7中引导适应性免疫应答。树突状细胞是最有效的抗原呈递的先天免疫系统的细胞;它们是专门用于摄取抗原，迁移到淋巴结，并激活幼稚CD4 +和细胞毒性CD8 + T细胞8-10。启用快速病原体检测的先天免疫系统利用众多种系编码的模式识别受体（PRR），识别保守的病原体衍生的基序或细胞应激和损伤的宿主衍生的标记。 Toll样受体（TLRs）是膜结合的模式识别受体识别某些胞吞噬病原体相关的分子模式（PAMP）和相关联的危险性分子模式（D-AMPS）。通过样受体（最接近现代种）对比点头是细胞质和的PAMP和阻尼的多元化作出回应。点头样受体重新目前抵御病原体逃避细胞表面和细胞内吞猪蓝耳病的第二行。病原体衍生的，或“危险”的相互作用有关，与TLR和NLR配体因素导致DC成熟而导致与其他免疫细胞和促进T细胞和自然杀伤细胞活化11增加直流互动的状态。 白细胞介素-1β是对感染的宿主防御的重要组成部分。在识别微生物，高促炎性细胞因子，IL-1β，分泌和功能的化学引诱剂和先天免疫和适应性免疫细胞激活剂， 在体内的IL-1β主要是负责对急性期反应包括发热和炎症细胞因子合成12。 最最接近现代种含有被认为在配体感应，中央核苷酸结合域（NACHT）是IMPO运作一个C端富含亮氨酸重复结构域rtant为NLRP3低​​聚和N末端效应器结构域（PYD在NLRP3），通过蛋白蛋白相互作用介导的信号转导至下游目标。在NLRP3蛋白质定义了最强烈的研究炎性复杂。这种蛋白质是NLR家族的成员，并具有以形成多分子蛋白复合物NLRP3，适配器蛋白PYCARD（也称为ASC）和ICE组成的能力。当炎性激活PYCARD结合NLRP3 N端结构域，并通过caspase活化和募集结构域（CARD）域新兵冰。白细胞介素-1转换酶最初产生为包含CARD的动机在其N-末端的酶原。炎性形成的结果使二ICE分子充分接近诱导其催化活性。炎性复合物是必要的激活ICE从而允许它来转换细胞质亲IL-1β成熟的细胞因子。 白细胞介素的分泌成功-1β在区议会需要检测两个不同的独立危险信号。首先，TLR传感的PAMP，DAMPS，或细胞因子信号传导（TNFα或IL-1β）的导致细胞质亲IL-1β蛋白表达的上调。第二，往往是不同的，信号所必需的炎性复合物的形成ICE成熟的上游。一些炎性刺激信号包括细菌膜孔形成毒素（如尼日利亚菌素），溶酶体破坏晶体（如单钠尿酸盐结晶，密歇根州立大学），和细胞外的ATP。上游机制领先了这些不同的激活剂，以NLRP3炎性体的激活目前还不清楚。调查研究炎性体形成的信令上游建议，细胞内的事件，如感应低钾血症或活性氧（ROS）的间接激活炎性13-28。 当中的NLRP3炎性体的不同的病毒活化是流感，它提供了bOTH所需的IL-1β分泌3，29-33初级和次级信号。使用鼠标NLRP3基因敲除模型发现，IL-1β分泌的DC是NLRP3依赖32。此外，NLRP3基因敲除小鼠引起的白细胞减少感染的部位和有经验的死亡率较高2，5。最近的两篇论文提出了一种机制，在流感病毒感染NLRP3炎性体的激活;首先，通过识别病毒RNA由TLR7或TLR8（取决于响应细胞的TLR表达）或通过传感共生细菌被其他的TLR诱导细胞质亲IL-1β的表达，随后的第二信号，激活NLRP3的吸反式高尔基网络33，34上形成炎性由病毒离子通道蛋白M2。在后者的步骤，触发NLRP3炎性体是由细胞内离子的干扰来实现&lt;em&gt;的环境导致ROS的产生，这是简单地说，通过NLPR3感觉到作为一个信号，以形成炎性。不过，ICE活性流感感染过程中炎性激活上游的确切机制仍不清楚。 这个工作描述了一种技术，用于研究NLRP3炎性体在人类moDCs可以用作用于通过一个很好的途径相关的DC基于响应于TLR8结扎IL-1β分泌与R848然后激活炎性体的进一步研究的基础有价值已知的激活NLRP3的，尼日利亚。这种方法的变体可以与其它细胞类型，包括可以使用，但不限于：单核细胞，巨噬细胞，以及其他DC亚群，和上皮细胞。

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			<td height="21" style="height:21px;width:391px;">Name of Reagent/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:121px;">Catalog Number</td>
			<td style="width:391px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">IL-4</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:391px;">GM-CSF</td>
			<td style="width:179px;">Genzyme</td>
			<td style="width:121px;">NDC 58468-0180-2</td>
			<td>We acquire this item through our local pharmacy with a prescription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">RPMI 1640 with L-glutamine</td>
			<td style="width:179px;">Cellgro</td>
			<td style="width:121px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Peripheral blood mononuclear cells</td>
			<td style="width:179px;">New York Blood Center</td>
			<td style="width:121px;"></td>
			<td>PBMCs were isolated from the blood of healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">12-well tissue culture plates</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">96-well round bottom tissue culture plates</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">3799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-IL-1&#946;-FITC</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;">IC201F</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">FITC isotype control</td>
			<td style="width:179px;">Miltenyi Biotec</td>
			<td style="width:121px;">130-092-213</td>
			<td></td>
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			<td height="21" style="height:21px;width:391px;">&#945;-&#946;-Tubulin</td>
			<td style="width:179px;">Santa Cruz</td>
			<td style="width:121px;">SC-9014</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-IL-1&#946;</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;">mab201</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:391px;">PVDF Immobilon-FL membrane</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:121px;">IPFL00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">gradient 4-12 % polycrylamide gel</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-1159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">laemmli sample buffer</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">BSA</td>
			<td style="width:179px;">Equitech Bio Inc</td>
			<td style="width:121px;"></td>
			<td>30% solution sterile/filtered</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">PFA</td>
			<td style="width:179px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">15710</td>
			<td>16% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">human inflammatory cytokine bead array kit</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">nigericin</td>
			<td style="width:179px;">Invivogen</td>
			<td>tlrl-nig</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">R848</td>
			<td>3M Corp.</td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-CD14</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">340436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-CD11c</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">555392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#946;-mercaptoethanol</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">TBS</td>
			<td style="width:179px;"></td>
			<td style="width:121px;"></td>
			<td>On site stock room</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tween-20</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">P2287-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a>Nunc EasYFlask 225cm2, Filter Cap, 70mL working volume, 30/Cs</a></td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">159933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">20 &#956;M Sterile Disposable Filter Units</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gentamicin</td>
			<td style="width:179px;">Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hepes</td>
			<td style="width:179px;">Invitrogen</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">goat &#945;-mouse IRDye 800CW</td>
			<td style="width:179px;">Licor</td>
			<td style="width:121px;">926-32210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">donkey &#945;-rabbit IRDye 680RD</td>
			<td style="width:179px;">Licor</td>
			<td style="width:121px;">926-68073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Spectra multicolor broad range protein ladder</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">26634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tris Glycine SDS 10x</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">1610732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tris Glycine 10x</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-0734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Methanol - 4L</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:121px;">A433P-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Prolong Gold antifade Reagent with DAPI</td>
			<td style="width:179px;">Life Technologies</td>
			<td>P-36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">8 chamber polystyrene vessel tissue culture treated glass slide</td>
			<td style="width:179px;">BD Falcon</td>
			<td style="width:121px;">354108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Poly-L-Lysine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:121px;">P4707</td>
			<td></td>
		</tr>
	</tbody>
</table>

activation and measurement of nlrp3 inflammasome activity using il-1&#946; in human monocyte-derived dendritic cells

从白细胞介素的分泌素（IL）产生的炎症过程-1家族细胞因子由免疫细胞引起局部或全身炎症反应，组织重塑和修复，以及病毒学控制1，2。白细胞介素-1β是先天免疫反应的基本要素，并有助于消除侵入的病原体，同时防止建立持续性感染1-5中。 Inflammasomes是关键信令平台白细胞介素1转化酶（ICE或胱天蛋白酶-1）的激活。在NLRP3炎性需要在DC中的至少两个信号，使IL-1β分泌6。亲IL-1β蛋白表达在静息细胞限制;因此，一个起动信号所必需的IL-1β的转录和蛋白表达的影响。第二信号中的多蛋白NLRP3炎性体的形成感测由NLRP3结果。树突状细胞，以respon的能力d，来所需的IL-1β分泌的信号可以使用合成嘌呤，R848，这是由TLR8在树突状细胞（moDCs），以素细胞人单核细胞检测到，随后激活NLRP3炎性体与细菌毒素进行试验和钾离子载体，尼日利亚。 单核细胞来源的DC很容易产生文化，并提供比纯化的人骨髓DC显著更多的细胞。这里介绍的方法不同于其他炎性测定法，它使用在体外人，而不是小鼠衍生的DC因此允许在人类疾病和感染的炎性体的研究。

这些技术测量TLR8与R848吸。细胞内细胞因子染色亲IL-1β允许从CD14显微镜和场向电流读数-的CD11c + moDCs。这两种技术都可以相对于定量给底漆的非，或休息，电池控制以及同种型对照（ 图1和2）。亲IL-1β+染色细胞的百分比乘以这一人群的几何平均，以提供平均荧光强度（MFI）。所述MFI为可比亲IL-1β存在于阳性染色的细胞的量。 免疫印迹措施...

Watch this Scientific Journal Video about Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1Î² in Human Monocyte-derived Dendritic Cells at JoVE.com

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

树突状细胞（DC）分泌IL-1β响应于TLR8识别合成嘌呤，R848，接着NLRP3炎性体激活与尼日利亚菌素，因此，IL-1β可以用来测量NLRP3炎性活性。细胞内细胞因子染色法，免疫印迹和ELISA法用于准确地测量通过IL-1β表达NLRP3炎性引发和活化。

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

activation-measurement-nlrp3-inflammasome-activity-using-il-1-human

Research

JoVE Journal

Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

20.7K 次观看.  New York University School of Medicine. 树突状细胞（DC）分泌IL-1β响应于TLR8识别合成嘌呤，R848，接着NLRP3炎性体激活与尼日利亚菌素，因此，IL-1β可以用来测量NLRP3炎性活性。细胞内细胞因子染色法，免疫印迹和ELISA法用于准确地测量通过IL-1β表达NLRP3炎性引发和活化。 

视频: Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

测量人体血浆中的凝血因子V活性，用酶联凝固分析

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

科研

免疫与感染

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

siRNA 电穿孔以在单纯疱疹病毒类型 1-感染单细胞衍生的丹突细胞中调制自噬

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...