这项研究是支持美国国立卫生研究院授予1K22AI097291-01和国家中心推进美国国立卫生研究院的转化科学奖下数UL1TR000433。我们也承认欧文达雷尔教授在麻省理工学院和斯蒂芬·马蒂亚斯在教授弗雷德·哈钦森癌症研究中心为他们的对疫苗的纳米粒子和OT-I /吕克·转基因小鼠的初步工作所作的贡献。

在本协议中描述的所有实验均按照既定的政策和准则经大学委员会使用与动物保健（UCUCA）在密歇根大学和执行。 1.合成和ICMVs表征合作富含蛋白质抗原和佐剂分子<ol><li>拌1:1,2- dioleoyl- SN -glycero -3-磷酸胆碱（DOPC）和1,2- dioleoyl- SN -glycero -3- phosphoethanolamine- -N 1摩尔比- [4-（对-maleimidophenyl）丁酰胺] （MPB）的氯仿，使总脂质含量以每批次1.26微摩尔（ 即 500微克DOPC和630微克MPB的）在20毫升玻璃小瓶中（直径= 28毫米，高度61例毫米）。 </li><li>加亲脂性药物，如单磷酰脂质A（MPLA），或亲脂性染料（ 例如，DID），以在所希望的浓度的脂质溶液。彻底额外干nitroge吹扫除去有机溶剂ñ气体并把样品在真空下O / N。 </li><li>通过加入200微升含有水溶性药物（ 如蛋白质抗原）的10mM的Bis-Tris丙烷（BTP，pH 7.0）中水合脂质膜。涡旋10秒，每10分钟进行1小时，在室温。 </li><li>转移从玻璃的内容小瓶入1.5ml微量离心管中，代替样品在冰水浴中，用40％强度在125瓦/ 20千赫探头尖端超声波仪的设置为5分钟，连续超声处理。 </li><li>加入4微升150mM的二硫苏糖醇（DTT）到各批次（工作浓度2.4毫摩尔），涡旋，并用台式微量离心机短暂离心。 </li><li>添加200mM的氯化钙 40微升并用吸管（工作浓度33毫摩尔）混合。孵育的样品在37℃下1小时，以允许MPB-含脂层用DTT的交联。 </li><li>离心样品以20,000×g离心15分钟，除去上清液，并重新悬浮于200μlddiH 2 O的</li><li>重复步骤1.7和离心后，再次在第二ddiH 2 O洗，从上清取出氯化钙2，未反应的DTT，和未封装的货物材料。 </li><li>在ddiH 2 O制备的2 kDa的聚乙二醇硫醇（PEG-SH）的10毫克/毫升重悬在100μl的PEG-SH溶液各ICMV样品和在37℃进行30分钟。 </li><li>执行两个ddiH 2 O洗涤（步骤1.7）和重悬最终ICMV沉淀在PBS和存储在4℃。在使用前，混合ICMV悬浮，作为粒子可以沉淀到长期储存后的底部。 </li><li>对于颗粒的表征，从每批除去ICMVs的一小等分试样（〜10％），并在1毫升ddiH 2 O的总体积为单独稀将单个样品用Zetasizer细胞和测量粒径，多分散性指数，并使用DLS和zeta电位测量系统（根据制造商的方案）样品的ζ电势。 </li></ol> 2.检查淋巴结排水与共聚焦显微镜荧光标记的ICMVs <ol><li>装载了荧光标记抗原和亲脂性荧光染料ICMVs的制备<ol><li>制备荧光标记的蛋白，如卵清蛋白的Alexa Fluor 555琥珀酰亚胺酯反应，可根据制造商的说明书。 </li><li>为了制备ICMVs具有标记的荧光团在脂质壳，添加亲脂性的荧光染料，（ 例如，1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- Tetramethylindodicarbocyanine，（DID））制备脂质膜的过程中（步骤1.2）以0.05％摩尔脂量。对于脂质膜水化（步骤1.3），含有荧光标记抗原使用缓冲区，并完成ICMV合成步骤1.4-1.11概述。 </li></ol></li><li>纳米颗粒在尾基部皮下给药<ol><li>使用受控流蒸发器配有感应腔ù麻醉小鼠根据一个IACUC tilizing 3％异氟烷和氧气流量为1.5升/分钟批准动物协议。一旦鼠标是无意识的，事先快速执行以下步骤来麻醉穿着胶制到注射部位最优访问，并尽量减少不适感的动物。另外，使用合适的鼻锥维持麻醉。如果小鼠麻醉的时间超过5分钟，应用必要眼润滑油以后的程序最小化刺激。 </li><li>喷用70％乙醇的尾根部消毒和润湿毛皮部分湿发，以暴露一小片皮肤可见，这可以被用于可视化在皮肤下针。 </li><li>制备含抗原和所需量的在PBS中，每100微升接种剂量的佐剂颗粒注射悬浮液（ 例如，10微克的OVA和每100微升注射剂量为0.3微克人运已经过去6,9使用）。 </li><li>绘制颗粒悬浮液INT办公自动化注射器用27-29ģ针和插入针头在尾巴的基部（约5毫米的发际线）与锥朝上，并注入50微升微粒的悬浮液22的。 </li><li>等待几秒钟的压力平衡，防​​止过度的回流并拔出针头出来。重复在尾巴基部的另一侧的注入同时针对排水腹股沟淋巴结。 </li></ol></li><li>淋巴结冰冻切片的制备和考试用激光共聚焦显微镜。 <ol><li>安乐死的小鼠用CO 2窒息，随后诱导气胸根据IACUC批准的动物协议。根据协议贝多亚23证明提取腹股沟淋巴结和洗出的血液通过将组织在1ml的4℃PBS。 </li><li>从与组织中的组织吸收的PBS并放置组织在组织cryomolds（10×10×5mm的3）预先填充顶端与华侨城冷冻介质24。捕捉冻结那朵在液氮告块30秒。或者，将组织块在干冰上30分钟。存储在-80冷冻组织℃冰箱。 </li><li>切的组织切片5-10微米厚的低温恒温器设定为-20℃的24。 </li><li>如果有必要，进行免疫荧光标记，并检查使用共焦显微镜组织如以前证明24。 </li></ol></li></ol>抗原特异性3.监测膨胀，萤光素酶表达的CD8 + T细胞的纳米接种所有的动物成像后<ol><li>的OVA 257-264特异性，从​​OT-I /吕克转基因小鼠的萤光素酶表达的CD8 + T细胞的分离<ol><li>安乐死的OT-I /吕克·转基因小鼠用CO 2窒息和诱导根据IACUC气胸批准的动物协议。通过访问腹膜腔和小心从胰腺23取下组织收获以无菌方式的脾，并地方在5ml 4℃PBS + 2％FBS中转移到组织培养罩。 </li><li>上放置一个70微米的尼龙过滤器的脾超过50毫升锥形离心管（最多3脾脏一次）。从3毫升注射器使用柱塞，通过过滤研磨细胞。 </li><li>清洗活塞，用PBS + 2％FBS过滤并丢弃。使总体积至10ml /脾脏中的50ml管中，以细胞悬浮液的一个小样品与血球计数，和离心机在300×g下10分钟。 </li><li>使用市售的磁性负选择的试剂盒，按照制造商的说明分离的CD8 + T细胞群。 </li><li>用PBS洗涤细胞后，计数分离的CD8 + T细胞的数量。为了评估所述分离的CD8 + T细胞的纯度，孵育〜20,000-30,000细胞在20μl小鼠CD16 / 32抗体（0.025毫克/毫升）的10分钟，然后加入20微升αCD8-APC抗体（0.005毫克/毫升）孵育30分钟。 PErform所有温育在4℃下在PBS + 1％w / v的BSA中。进行流式细胞仪分析25。 </li></ol></li><li>孤立的CD8 + T细胞过继转移他们的扩张接种疫苗后的可视化和<ol><li>通过在PBS中的经静脉内尾静脉注射22（-1天）200微升体积施用1-10×10 5个细胞进行过继转移分离的OT-I /吕克CD8 + T细胞的成幼稚C57BL / 6小鼠。考虑到C57BL / 6小鼠皮毛和皮肤黑补丁可以与生物发光信号干扰，剃光白化C57BL / 6小鼠非常适合于这些研究。 </li><li>在一天（第0天）后，施用疫苗如前所述（第2.2节）。 </li><li>施用150毫克每千克小鼠体重萤光素腹膜内的PBS 300微升体积。 10分钟后，麻醉小鼠用异氟烷（在步骤2.2.1）和可视化的OT-I /吕克CD8 + T细胞通过获取生物发光信号5-10分钟瓦特第i个整体动物成像系统（IVIS;参照威尔逊26详细说明）。必要时重复进行纵向研究。 </li></ol></li></ol>的PBMC 4.肽的MHC四聚体染色进行分析的抗原特异性的CD8 + T细胞注意:下面的协议程序可以使用具有的OT-I /吕克CD8 + T细胞或C57BL / 6小鼠过继转移而不继转移或者C57BL / 6小鼠中进行。 <ol><li>在接种后的所需时间点，收集约100微升的血液（4-6滴）从小鼠经由颌下出血技术27到涂敷用K 2 EDTA的管中，颠倒几次，以防止凝固。 </li><li>转移100微升血液到离心管中，加入1毫升的裂解缓冲液中，以去除红血细胞（红细胞）孵育2至3分钟。离心样品5分钟，在1500×g离心，去除上清。如果仍沉淀一ppears红色（表示不完全去除红细胞），重复裂解步骤用的裂解缓冲液的简要孵育（&lt;1分钟）。 </li><li>用1ml的FACS缓冲液（PBS + 1％w / v的BSA）和离心机在1500×g离心洗剩余的PBMC 5分钟。 </li><li>吸出上清，悬浮于20μl小鼠CD16 / 32抗体的样品（0.025毫克/毫升），以阻断非特异性和的FcR介导的抗体结合。孵育在室温下10分钟。 </li><li>从离心管转移细胞分为4毫升圆底流式细胞仪管。加入20微升的H-2K的b OVA四聚-SIINFEKL-PE溶液根据制造商的规格，以每个样品孵育在冰上30分钟。 </li><li>制备抗体混合物（ 例如，αCD8-APC，αCD44-FITC，并且αCD62L-PECy7抗体（0.005，0.005，和0.002毫克/毫升浓度，分别））。加入20微升的每个实验样品，孵育在冰上20分钟。通过准备拉单荧光控制乱棍细胞在上面指出的浓度各荧光标记的四聚体或抗体。 </li><li>洗2次用FACS缓冲液重悬在含有DAPI的2微克/毫升FACS缓冲液的最终的沉淀。这些细胞现在已经准备好了流式细胞分析（细节和例子可以Scheffold 25找到）。 </li></ol>

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本文中提供的协议说明了合成和一个新的基于脂质的纳米颗粒系统的表征，称为ICMVs，并提供验证的基于纳米颗粒的疫苗制剂的有效性，以诱导抗原特异性CD8 + T细胞应答的过程。 ICMV合成以全含水的条件，这是一个重大的优点与其他常用的聚合物纳米颗粒系统（ 例如 ，聚（丙交酯-共-乙）酸颗粒），通常需要为制备有机溶剂中，经常造成的损失相比，完成在抗原蛋白质抗原29,30。此...

传统疫苗的发展主要采用试错的经验方法。但是，与生物材料和发现免疫激活的分子决定因素的各种各样的最近发展，现在有可能合理设计疫苗制剂与来自病原体1,2-衍生生物物理和生物化学的线索。特别地，各种微粒药物递送平台已经被检查的，因为它们可以被共装载亚基的抗原和免疫刺激剂的疫苗载体，保护疫苗成分免于降解，并提高它们的共同递送至抗原呈递细胞（APC）存在于淋巴节点（逻辑节点），从而最大限度地提高免疫刺激和活化3-5。在这份报告中，我们描述了"病原体模仿"纳米颗粒系统的合成，称为interbilayer交联多层囊泡（ICMVs），先前已经被证明是一种有效的疫苗platfor米为健壮的细胞毒性T淋巴细胞（CTL）和在全身和粘膜组织区室6-9体液免疫应答诱导。特别是，接种ICMVs取得了实质性的提高血清IgG水平对疟疾抗原，与接种常规助剂（ 如明矾和Montanide中）7相比，也引发对肿瘤细胞和小鼠9病毒攻击模式有效的CTL反应。在这里，用ICMVs作为模型疫苗纳米粒子系统，我们描述的方法对疫苗的纳米制剂特性，包括粒径和zeta电位测量和粒子贩卖跟踪，以引流淋巴结（DLNS）利用冰冻切片组织7的共聚焦成像。此外，我们提出了荧光素酶表达的抗原特异性CD8 + T细胞9,10继转移后分析在小鼠中膨胀CTL反应的全动物基于成像的方法。最后，我们去外周血单核细胞（PBMC），用于在接种纳米粒子6,9-小鼠内源性T细胞应答纵向量化的划线四聚体染色。 ICMVs是一个基于脂质的纳米颗粒制剂通过简单的脂质体受控聚变合成为多层结构，然后将其化学地交联的马来酰亚胺官能化的磷脂头部基团稳定化的内脂质层与二硫酚交联剂6。一旦ICMVs合成中，纳米粒子的一小部分可以被用于确定颗粒尺寸和ζ电势（ 即，表面的粒子的电荷）与动态光散射（DLS）系统和ζ电位分析仪。 DLS措施中的光散射颗粒悬浮液的变化，允许确定扩散系数和粒子11的流体动力学大小。获得一致的粒径不同批次的合成是关键由于粒径是通过的APC 12,13影响疫苗颗粒的淋巴引流到DLNS和随后的细胞摄取的主要因素之一。此外，ζ电势可以通过测量当电流被施加在粒子速度，这允许确定颗粒和颗粒表面电荷11的电泳迁移率来获得。确保颗粒一致ζ电位值是重要的，因为颗粒表面电荷确定胶体稳定性，其具有在储存过程中和在体内给药14,15后在颗粒分散体直接影响。为了跟踪的粒子定位到DLNS，ICMVs可以带有所需荧光团包括亲脂性染料和共价标记的抗原。免疫接种后，小鼠可以在不同时间点进行安乐死，DLNS切除，冰冻切片，并用共聚焦显微镜分析。这种技术允许淋巴的DraⅠ可视化宁纳米颗粒疫苗载体和抗原DLNS两者。组织切片可另外被染色用荧光标记的抗体和用于获得详细信息，诸如种类与抗原和形成生发中心有关的细胞，因为我们先前7所示。 一旦粒子合成进行了优化，并贩卖给DLNS一经确认，以验证在体内 CTL扩张启发是非常重要的。为了分析抗原特异性CD8 + T细胞的诱导响应于纳米颗粒疫苗，我们已利用模型抗原，卵白蛋白（OVA），以OVA 257-264肽（SIINFEKL）免疫显性CD8 + T细胞表位，它允许详细的免疫学分析对于最初疫苗开发16,17抗原特异性T细胞应答。特别是，以询问膨胀和抗原特异性CD8 + T细胞的迁移的动力学，我们已经产生了双转基因小鼠模型通过杂交萤火虫荧光素酶表达的转基因小鼠（吕克）与OT-I转基因小鼠具有CD8 + T细胞与T细胞受体（TCR）特异性针对SIINFEKL（在用H-2K B缔合 ）。从这些的OT-I /吕克小鼠，荧光素酶表达，OT-I CD8 + T细胞可以被分离并用于过继转移进入幼稚C57BL / 6小鼠制备。一旦晶种，成功免疫用含有OVA纳米颗粒将导致扩张的转移的T细胞，其可以通过与一个整体动物成像系统9,10监测的生物发光信号来跟踪的。这种非侵入性全身成像技术已用于与其他的病毒或肿瘤抗原，在过去18至20，露出参与T细胞扩增在淋巴组织和传播至外周组织中的纵向的方式处理。 互补继转移抗原特异性CD8 + T细胞的分析，endogeno我们的T细胞应答接种后可以检查与肽-主要组织相容性复合体（MHC）四聚体测定21，其中一个肽-MHC四聚体复合物，由四个荧光团标记的MHC-I类分子装载肽表位，采用结合TCR和标签CD8 + T细胞以抗原特异性方式。肽的MHC四聚体分析可以在终端尸检研究，以确定抗原特异性CD8 + T细胞在淋巴和外周组织或与来自串行抽血获得外周血单核细胞（PBMC）的纵向研究来进行。染色的淋巴细胞与肽-MHC四聚物之后，流式细胞仪分析，执行对CTL的CD8 + T细胞中的频率的表型或定量的详细分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>870012</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>850375</td>
			<td></td>
		</tr>
		<tr>
			<td>Monophosphoryl Lipid A (Synthetic) (PHAD&#8482;) (MPLA)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>699800</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vials</td>
			<td>Wheaton</td>
			<td>0334125D</td>
			<td></td>
		</tr>
		<tr>
			<td>Symphny Vacuum Oven</td>
			<td>VWR</td>
			<td>414004-580</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin (OVA)</td>
			<td>Worthington</td>
			<td>3054</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-Tris Propane (BTP)</td>
			<td>Fisher</td>
			<td>BP2943</td>
			<td></td>
		</tr>
		<tr>
			<td>Q125 Sonicator (125W/20kHz)</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Fisher</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>2 kDa Thiolated Polyethylene Glycol (PEG-SH)</td>
			<td>Laysan Bio</td>
			<td>MPEG-SH-2000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern ZetaSizer Nano ZSP&#160;</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer Cuvettes</td>
			<td>Malvern</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID)</td>
			<td>Life Technologies</td>
			<td>D-7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-succinimidyl ester (AF555-NHS)</td>
			<td>Life Technologies</td>
			<td>A37571</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT freezing medium&#160;</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cryomolds</td>
			<td>VWR</td>
			<td>25608-922</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Albino C57BL/6 mice</td>
			<td>Jackson</td>
			<td>000058</td>
			<td></td>
		</tr>
		<tr>
			<td>OT-1 C57BL/6 mice</td>
			<td>Jackson</td>
			<td>003831</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon strainer</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep&#8482; Mouse CD8+ T Cell Isolation Kit</td>
			<td>StemCell</td>
			<td>19853</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS&#174; whole animal imaging system</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2EDTA tubes</td>
			<td>BD</td>
			<td>365974</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 Fc Block</td>
			<td>Ebioscience</td>
			<td>14-0161-86</td>
			<td></td>
		</tr>
		<tr>
			<td>H-2Kb OVA Tetramer</td>
			<td>MBL</td>
			<td>TS-5001-1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8-APC</td>
			<td>BD</td>
			<td>553031</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD44-FITC</td>
			<td>BD</td>
			<td>553133</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD62L-PECy7</td>
			<td>Ebioscience</td>
			<td>25-0621-82</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>SIGMA</td>
			<td>D8417-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>CyAn Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-animal imaging and flow cytometric techniques for analysis of antigen-specific cd8+ t cell responses after nanoparticle vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

参与ICMVs的合成步骤示于图1 6。简言之，将包含任何亲脂性药物或荧光染料的脂质膜水合的亲水性药物的存在。二价阳离子，如Ca 2+，加到驱动聚变阴离子脂质体成多层囊泡。二硫醇交联剂，诸如DTT，被添加到"钉"马来酰亚胺官能化的脂质上并置脂质层，最后剩余的外部马来酰亚胺基团被淬灭与硫醇化-PEG部分的反应。每批的一小部分可以通过确定粒径，多分散性指数，...

Watch this Scientific Journal Video about Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination at JoVE.com

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

whole-animal-imaging-flow-cytometric-techniques-for-analysis-antigen

Research

JoVE Journal

Immunology and Infection

13.2K 次观看.  University of Michigan. We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

视频: Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

枯否细胞分离的纳米粒子毒性测试

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

科研

免疫与感染

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...