作者感谢帕特里夏巴达西，凡妮莎Lagal和萨宾Thiberge为批判性阅读，伊琳娜·多布雷斯库和萨宾Thiberge的帮助在小鼠皮肤拍照和Pauline Formaglio教学中的子孢子蠕动体内成像。我们还要感谢马立克Szatanik和中心的生产和按蚊（CEPIA - 巴斯德研究所）感染蚊子饲养。这项研究是由安盛研究基金和基金从科特迪瓦了Laboratoire卓越"新发传染性疾病的综合生物学"（批准号。ANR-10的LabX-62-IBEID）。

<html>所有的程序是由巴斯德研究所的委员会，由当地伦理委员会对动物实验（伦理委员会IDF-1巴黎，巴黎，法国，协议编号:2012-0015）批准，并按照适用的原则和规定执行。 1.材料与试剂<ol><li>使用对感染小鼠产生和后部后3-5天喂如前面所述21雌性按蚊的蚊子（Sda500株）。 </li><li>使用寄生虫疟原虫 ANKA克隆表达组成型热休克蛋白70（HSP70）启动子的控制下的绿色荧光蛋白（GFP）基因。这将产生在整个寄生虫生命周期的22明亮的荧光。 </li><li>使用雌性C57BL / 6JRj小鼠（7周龄）。 注:所描述的协议中使用的所有试剂都列在T 能材料/设备 。 </li></ol>_title"&gt; 2，辐射衰减蚊虫唾液腺子孢子隔离<ol><li>之间的传染性血粉后18-25天，收集并如先前21描述的冷麻醉蚊子成在冰上15毫升管。小心通过倒转该管将它们转移到培养皿。保持冰虫和揭露蚊子γ-或X线照射的12拉德剂量。 </li><li>从辐照蚊虫唾液腺隔离减毒子孢子先前所描述的21。收集感染唾液腺在冰上1×Dulbecco氏磷酸盐缓冲盐水（DPBS）中的一小体积（大约15微升），并轻轻粉碎他们释放子孢子。 注意:高度浓缩的寄生虫悬浮液中的小体积为临界的注射，这是因此，必须收获足够数量的唾液腺从大量预选良好感染蚊子，其稳健的表达所证明绿色荧光。 </li><li>通过35微米的细胞过滤帽，以消除团块和蚊子碎片适于一个低粘附力1.5 ml离心管过滤孢子悬浮液。 </li><li>算孤立子孢子总数如前所述21，用冷的1×DPBS调整寄生虫悬浮液的浓度，以获得83000孢子/微升。存储寄生虫冰同时继续下一步骤。 </li><li>收集来自未感染的蚊子唾液腺将用作阴性对照和作为在步骤2.2和2.3中所述处理该样品的当量数。稀如上所述，得到在悬浮唾液腺提取物的类似浓度的样品。 注:子孢子可以存储在冰上最多2小时。然而，理想情况下他们是在一个小时内破碎唾液腺后注射。 </li></ol> 3.注射孢子进入皮肤拉耳揭掉<ol><li>通过腹腔注射氯胺酮（50毫克/千克）/赛拉嗪（5mg / kg的）混合物麻醉动物。等待5-8分钟的鼠标是在无意识的状态和应用眼药膏的眼睛，以防止角膜干燥。通过两个后双脚进行温和的脚趾捏评价麻醉深度。 注:麻醉的剂量持续少于20分钟，因此，下一个步骤必须迅速完成。 </li><li>通过固定与先前立体显微镜（ 图1A）根据放置的透明胶带腹侧稳定鼠标的耳廓。轻压耳朵，避免血管损伤。 </li><li>装入10微升注射器/ 35计斜面针与0.6微升悬浮寄生虫10。 </li><li>通过仔细插入在耳朵上（ 图1A）的背部侧的针，表皮下面交付子孢子悬浮液在4注射部位（每个站点0.15微升），用斜面向上，注意避免血管。允许针保持在真皮为几秒钟，以防止注入剂的回流后，再取出。在步骤（ 图1B和1C）的端部观察在每个注射部位的特性丘疹。 </li><li>注射后，小心地取出从磁带的耳朵。 </li><li>由以下的步骤3.2-3.4注射对侧耳朵与相同剂量的寄生虫。 </li><li>注入2用1×DPBS任0.6微升或0.6微升未感染的蚊子1X DPBS +唾液腺提取物的额外的小鼠，以评价由单独的注入和蚊子材料在真皮沉积介导的炎症反应。 </li><li>把它放回笼子前，监视鼠标麻醉复苏。 注:在皮肤上的适当的子孢子沉积可通过荧光显微镜（ 图2A和2B进行监测），如previsously 23所述正在编写的鼠标。 </li></ol> 4.皮肤和引流淋巴结清扫术<ol><li>制备标准培养基（SM）如下:Dulbecco改良的Eagle培养基（DMEM）+ 25毫4-（2-羟乙基）补充有400单位-1-哌嗪乙磺酸（HEPES）/ ml胶原酶和50微克/毫升脱氧核糖核酸酶（DNA酶） 。 </li><li>准备在冰两个6孔平底组织培养板用4.5ml SM + 70微米的细胞过滤每孔DLN样品（板甲）和每孔4.5毫升SM皮肤样本（板B）。 </li><li>在以下的子孢子接种所需时间点，深受腹腔注射氯胺酮（125毫克/千克）/赛拉嗪（12.5毫克/千克）混合物之前颈脱位麻醉小鼠。确保鼠标演示临床死亡的迹象，并立即开始清扫。 注:骨髓细胞招聘的动力学可以在注射后的不同时间进行检查挠度（ 即 2小时，4小时，并如前所述14 24小时）。 </li><li>消毒接种耳并用70％乙醇的相应颈部区域，以减少污染的可能性。 </li><li>用无菌剪刀和镊子，无菌解剖近端耳DLN而不收集周围的脂肪。请在jugulo-carotidian沟第一切口，然后从工作现场取下毛发。伸展用2钳子切口以暴露相比周围组织出现灰色的DLN。 </li><li>小心捏在DLN的顶部钳结缔组织，以促进其除去。通过将钳子的淋巴组织（ 图3A和3B）的下方提取DLN。放置在DLN井含4.5毫升SM + 70微米的细胞过滤（板甲）。 </li><li>使用无菌锋利的剪刀和镊子（ 图4A）收获整个接种耳朵。 SEPAR捏和间隔开切两钳（ - 4E 图4B）结束吃耳朵的背部和腹部的方面。它们放到一个很好含4.5毫升SM（板B），确保这两个组织都完全沉浸在网上平台。 注意:为了提高实验的可靠性和增加所关注的细胞的数目，池2注入的耳朵或2 DLNS从每个样品相同的鼠标。 </li><li>收获和并行处理的耳朵和与任一1X DPBS或唾液腺提取物接种的小鼠的DLNS。包括从幼稚鼠标负，单染补偿控制收获2个额外的耳朵和淋巴结样品。 </li></ol> 5.淋巴结细胞分离<ol><li>在37℃+ 5％CO 2孵育淋巴结（图版A），参考下缓慢搅拌15分钟。添加10mM的最终乙二胺四乙酸（EDTA），每孔中和胶原酶和DNA酶ACTIvities。进行冰下一个步骤。 </li><li>分离使用5毫升注射器柱塞的顶端淋巴结。按在对阵过滤，直到所有剩下的打圈方式是白色的结缔组织。 </li><li>用500μl1X DPBS + 1％胎牛血清（FBS）+ 2mM的EDTA冲洗细胞过滤两次。转移阱的整个体积在15ml管中。 </li><li>冲洗用500μl1X DPBS + 2％FBS + 2mM的EDTA的阱两次，并且将体积转移到15毫升管。置于冰上管。 </li></ol> 6.从耳朵皮肤细胞分离<ol><li>在37℃+ 5％CO 2孵育耳小叶（图版B）为下温和搅拌1小时。 注:温育20分钟后，切穗小叶用剪刀切成小块，以促进组织消化，并把样品放回培养箱剩余的40分钟。所用的介质在温育时间结束时可以观察到单个的细胞。 </li><li>添加10毫米最后<html>每孔EDTA来中和胶原酶和DNA酶的活动。进行冰下一个步骤。 </li><li>传送中使用1ml的吸管新井含有70微米的细胞过滤的耳组织碎片和介质的整个体积。从尖端的端部切成2〜3毫米更容易地收集耳组织碎片。 </li><li>分离使用5毫升注射器柱塞的顶端耳组织碎片。按在对阵过滤，直到所有剩下的打圈方式是白色的结缔组织。 </li><li>用500μl1X DPBS + 2％FBS + 2毫米EDTA冲洗过滤单元的两倍。 </li><li>通过一个70微米的细胞滤网转移井的整个体积到50ml管。 </li><li>冲洗孔用500μl1X DPBS + 2％FBS + 2mM EDTA的和体积转移到50毫升管。保持管冰。 注意:不同的步骤来的总细胞从皮肤分离和DLN组织总结于图5。 </li></ol>标题"&gt; 7。数量和收集的细胞活力<ol><li>离心皮肤和DLN样品5分钟，在450 xg离心在4℃下，并丢弃上清液。轻轻地用300微升1X DPBS + 2％FBS + 2毫米EDTA悬浮颗粒。 </li><li>收集每个样品的一个小体积，以计数从使用血细胞计数器对皮肤和DLN分离的细胞的总数。添加0.04％台盼蓝以确定细胞活力。 </li></ol> 8.阻断非抗原特异性结合<ol><li>加入10微克/毫升未标记CD16 / CD32的Fc块的抗体。孵育20分钟，在4°C（可以去更长）。 </li><li>加入2毫升冷1X DPBS + 2％FBS + 2毫摩尔EDTA的。离心样品5分钟，在4℃450 XG并弃去上清液。轻轻重悬沉淀用300μl1X DPBS + 2％FBS + 2mM EDTA的并保持在冰上的样品。 </li></ol> 9.染色皮肤和淋巴结的髓系细胞<ol><li>此前孵育滑雪受阻n和DLN分离活/死跟踪（ 即 4&#39;，6-二脒基-2-苯基或DAPI），抗CD45和抗CD11b特异性抗体的细胞。 注意:为了丰富感兴趣的髓样细胞亚群，尤其是稀有的人，CD45 +细胞和淋巴细胞可分别从皮肤纯化或通过磁珠促进细胞分选从DLN耗尽。因为大多数从DLN中分离的细胞是CD45 +，使用抗CD45的不是强制性的。其他标记物可以被包括在由正或负门控策略来识别感兴趣的髓样细胞亚群。在这个协议中，在DLN招募的骨髓细胞，通过排除CD8α+细胞富集的（全T细胞隔室可通过使用抗CD3特异性抗体靶向）。 </li><li>在黑暗中孵育30分钟，在4℃下。在450 XG加入500μl1X DPBS + 2％FBS + 2mM EDTA的，离心样品5分钟，在4℃并丢弃苏佩rnatant。重复洗涤步骤两次。 </li><li>重悬用300μl1X DPBS + 2％FBS + 2毫米EDTA样本。通过35微米的细胞滤网传送整个体积中在FACS管中。冲洗用100微升1X DPBS + 2％FBS + 2毫米EDTA过滤器。 </li><li>运行在一个流式细胞染色细胞。门活的单细胞（DAPI - ，FSC-W）排除死细胞和双峰。情节的 CD45 +细胞CD11b +事件对皮肤（ 图6A）和（CD45 +）CD8α -细胞CD11b +用于DLN（ 图6B）的事件。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+capture+of+infected+neutrophils+by+dendritic+cells+in+the+skin+inhibits+the+early+anti-leishmania+response.">Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response.</a> PLoS Pathog. 8 (2), e1002536 (2012).</li><li>Thiberge, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo.+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo. imaging of malaria parasites in the murine liver.</a> Nat Protoc. 2 (7), 1811-1818 (2007).</li><li>Ishino, T., Orito, Y., Chinzei, Y., Yuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-dependent+protein+kinase+regulates+Plasmodium+ookinete+access+to+the+midgut+epithelial+cell.">A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell.</a> Mol Microbiol. 59 (4), 1175-1184 (2006).</li><li>Amino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+malaria+sporozoites+in+the+dermis+of+the+mammalian+host.">Imaging malaria sporozoites in the dermis of the mammalian host.</a> Nat Protoc. 2 (7), 1705-1712 (2007).</li><li>Ploemen, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+liver+load+following+parenteral+sporozoite+administration+in+rodents.">Plasmodium liver load following parenteral sporozoite administration in rodents.</a> Vaccine. 31 (34), 3410-3416 (2013).</li><li>Epstein, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+malaria+vaccine+designed+to+protect+through+hepatic+CD8+T+cell+immunity.">Live attenuated malaria vaccine designed to protect through hepatic CD8 T cell immunity.</a> Science. 334 (6055), 475-480 (2011).</li><li>Roestenberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+protection+against+malaria+after+experimental+sporozoite+inoculation:+an+open-label+follow-up+study.">Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.</a> Lancet. 377 (9779), 1770-1776 (2011).</li><li>Seder, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+malaria+by+intravenous+immunization+with+a+nonreplicating+sporozoite+vaccine.">Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.</a> Science. 341 (6152), 1359-1365 (2013).</li><li>Douradinha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+attenuated+P36p-deficient+Plasmodium+berghei+sporozoites+confer+long-lasting+and+partial+cross-species+protection.">Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection.</a> Int J Parasitol. 37 (13), 1511-1519 (2007).</li><li>Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Dendritic+Cells+and+Macrophages+from+the+Mouse+Intestine.">Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine.</a> J Vis Exp. (63), e4040 (2012).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> Eur J Microbiol Immunol (Bp. 2 (2), 112-120 (2012).</li></ol>

作者宣称，他们没有竞争的经济利益。

在使用全子孢子疟疾疫苗的人类大规模疫苗接种的角度来看，要克服的主要挑战之一是开发优化的路线和寄生虫的给药方法，以确保成功的免疫和保护24,25。在人类中，由减毒活寄生虫（LAP）介导的保护效力的评估具有以下自然蚊子被执行叮咬2，以及皮内，皮下25,26和IV免疫27。如在啮齿类动物28和非人灵长类25报道，LAP的IV施用相比，上述路线诱导?...

疟疾是最致命的传染病在世界上，造成每年超过五十万的人。感染疟原虫 ，该疾病的致病因子，开始由预红细胞（PE）的阶段。在这个阶段中，注入到由阴按蚊宿主皮肤的子孢子达到通过血流肝脏和分化内肝细胞成感染红血细胞，并导致疾病的症状的寄生虫的形式。 疟原虫的PE阶段代表了抗疟疾疫苗特权的目标。事实上，对住这些阶段减毒疫苗，如辐射衰减孢子（RAS），基因逮捕寄生虫（GAP）或药物预防和孢子（CPS）已经证明他们保护啮齿类和人类宿主1-9的能力。在啮齿动物模型，大多数疫苗研究是利用进行静脉免疫，这是在保护效力方面的黄金标准。然而，皮肤阶段的描述，并在引发保护皮肤相关引流淋巴结（DLN）的重要性，已经改变了我们的PE阶段的感知，并强调喷射的皮内途径的重要性。 P的活体成像注入啮齿动物的皮肤疟原虫子孢子已经表明，只有〜25％的接种的到达经由血流肝脏。剩余的〜75％的基DLN（〜15％）和皮肤（〜50％）10,11，其中一小部分可以皮肤细胞12,13内变换和仍然存活数周之间分配。此外，随后的研究描述了皮内免疫后建立有效的保护性免疫的主要发生在皮肤-DLN，其中寄生虫特异性CD8 + T细胞被激活的地方，并且仅在脾或肝DLNS 14,15轻微。 而大多数研究集中于在建立保护性免疫应答的牵连的效应细胞的表征，较少被知道关于注射进皮肤活的减毒的寄生虫的命运，特别是它们的先天免疫系统的相互作用。特别是，涉及在寄生虫抗原摄取，加工和呈递至CD8 + T细胞的抗原呈递细胞的表征是至关重要的，因为知道PE抗原采集既可以在皮肤和DLN隔间发生。上一页活体成像的研究以下感染蚊子叮咬16，而在DLN 10,17观察孢子和树突状细胞之间的相互作用早在肌肤明亮描述荧光赖氨酸-GFP阳性细胞的早期涌入。最近，已经报道，在由蚊子皮肤接种孢子同时增加树突和调节性T细胞的运动中的皮肤小鼠中，而在DLN 18观察抗原呈递细胞的减少数量。 我们的目的是确定和量化更精确地招募在皮肤和相应DLN以及那些以下免疫剂量的RAS 19的皮内注射与寄生虫相互作用的白细胞亚群。在这方面，我们分离出两种组织的骨髓细胞（CD45 +细胞CD11b +）和由多=参数流特征的感兴趣的亚群术。在利什曼原虫皮肤感染20的早期阶段中描述的免疫应答相一致，主主机响应于子孢子注射由随后的炎性单核细胞（CD45 +细胞CD11b中性粒细胞（CD45 +细胞CD11b + Ly6G +的Ly6C INT）的一个连续招募+ Ly6G -的Ly6C +），这些differenti的基础上确定的在Ly6G和的Ly6C表面标记的人表达。 我们在这里描述用于从小鼠皮肤中分离的骨髓细胞和以下免疫从感染蚊唾液腺提取剂量的RAS的皮内注射DLN的协议。可重复的皮内注射和组织处理的量化感染的组织内浸润的细胞群的表型变化的关键步骤。下面详述的方法提供了一种可靠的测定法来评估皮肤和DLN炎性响应于疟原虫寄生虫和可扩展到各种实验系统。

<table>
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C5138</td>
			<td>400 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain (0,4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Dilution 1:10&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32 (2.4G2 clone)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>10&#181;g/ml final (1:50)</td>
		</tr>
		<tr>
			<td>DAPI FluoroPureTM grade</td>
			<td>Life Technologies</td>
			<td>D21490</td>
			<td>1&#181;g/ml final</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (30-F11 clone)</td>
			<td>BD Biosciences</td>
			<td>559864</td>
			<td>&#160;Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (M1/70 clone)</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td>&#160;Dilution 1:400</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945; (5H10 clone)</td>
			<td>Life Technologies</td>
			<td>MCD0830</td>
			<td>&#160;Dilution 1:100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)&#160;</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

myeloid cell isolation from mouse skin and draining lymph node following intradermal immunization with live attenuated plasmodium sporozoites

当疟原虫的子孢子阶段接种到哺乳动物宿主的通过蚊子叮咬皮肤疟疾感染开始。高度能动的寄生虫不仅到达肝脏侵入肝细胞转化为红细胞感染形式。它也迁移到皮肤和引流注射部位，在那里它可以被识别和居民和/或招募骨髓细胞退化近侧淋巴结。活体成像报道在皮肤发明亮荧光的赖氨酸GFP阳性白细胞和在引流淋巴结的子孢子和的CD11c +细胞之间的相互作用的早期募集。在这里，我们提出了一个有效的步骤来恢复，识别和枚举招募到小鼠皮肤和引流淋巴结继小鼠模型免疫剂量子孢子的皮内注射骨髓细胞亚群。表型特征采用多参数流式细胞仪提供一个可靠的检测，以评估疟原虫感染的炎症反应在早期的动态细胞变化。

我们最近表现出免疫剂量P的那针注射器注射疟原虫子孢子在小鼠皮肤诱导中性粒细胞，随后通过在皮肤和DLN 19炎性单核细胞的连续募集。该议定书部分所述上面详细说明了用于成功地分离从两个组织以下的在耳真皮大量孢子的多次注射活髓样细胞的方法（图1和2）。内的第一个24小时后，将DLN（图3）与皮肤注射?...

Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

myeloid-cell-isolation-from-mouse-skin-draining-lymph-node-following

Research

JoVE Journal

Immunology and Infection

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

17.1K 次观看.  Institut Pasteur. We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

视频: Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

小鼠浅表淋巴结手术

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...