NGL是由产妇，围产期和儿童健康的韦恩州立大学围产期倡议的支持。我们非常感谢莫林McGerty和Amy E. Furcron（韦恩州立大学），他们的手稿的读数。

与之前在此协议中提到的样品工作，动物伦理委员会批准，必须由当地研究伦理委员会和机构审查委员会给出。当与动物血，细胞，或如本协议的有害因素的工作，适当的生物安全和实验室安全的行动必须遵循的。 1.鼠标操作和组织收集<ol><li>准备一个无菌工作站，并获得无菌工具组织收集。这些工具将包括大型和小型手术剪，钳，细尖镊子。包括标记有相应的组织名称小培养皿，填充有1×磷酸盐缓冲盐水（PBS）溶液（3 - 5ml）中平衡至室温（20 - 22℃）。还包括一个大培养皿，未标记的，填充用1×PBS溶液（10 - 15毫升）中。 </li><li>安乐死一个孕鼠（10.5至19.0天后交配（DPC）或交付前）使用二氧化碳（CO 2）。以确保该鼠标安乐死，使用颈椎脱位技术。 注:在10.5 DPC，则难以手动从子宫组织中分离的蜕膜组织。如果不需要蜕膜白细胞的分离，从非妊娠小鼠的子宫组织或那些来自小鼠白细胞&lt;10.5 DPC也可以通过以下这种协议进行的。 </li><li>使用一对无菌手术剪刀，彻底去除皮肤和肌肉组织的下腹部。用钳子，将抛开一切其他器官，直到子宫和卵巢是可见的。与子宫还连着，使用镊子将其移动腹膜腔（ 图1A）的外部。 </li><li>定位远端每个子宫角的输卵管和uterotubal结卵巢。使用小手术剪，做一个切口在uterotubal交界处，分离含有子宫血管肠系膜子宫角;那么，切除在子宫颈分离从腹膜腔（ 图1B）的子宫角。 </li><li>与小手术剪，使子宫颈和子宫角的下段之间的切口。休假在此过程中（ 图1C）附子宫颈的一部分，以避免打开子宫角。 </li><li>浸入子宫角（包括子宫颈）在一个大培养皿填充用1×PBS溶液（10 - 15毫升）中，以水化植入位置。 </li><li>虽然还沉浸在1X PBS液，修剪任何脂肪从子宫角与小手术剪。保持浸渍在整个解剖的1×PBS溶液中的组织。 </li><li>保持子宫角的地方与镊子，除去从通过间注入区切的横向子宫植入位点之一，用小的外科剪刀（ 图1D）。植入部位包括胎儿，被包围绒毛尿囊膜，和子宫，蜕膜和胎盘组织。 </li><li>使用镊子，保持现场到位并作一小切口在相邻的胎盘和蜕膜（ 图1E）在子宫壁。修剪周围胎盘/蜕膜的周边，直到这些来自两个子宫壁和绒毛尿囊膜，其包括胎儿（ 图1F）变得分离。 </li><li>取出胎盘和连接蜕膜组织和地方1X PBS液（3 - - 5毫升，见步骤1.12）。 </li><li>放置由附着在子宫组织在培养皿中填充用1×PBS溶液中的绒毛尿囊膜包围的胎儿（3 - - 5毫升）中。这些组织的水合将促进从子宫壁的绒毛尿囊膜的分离（见步骤1.14）。 </li><li>用一双细尖镊子，轻轻剥离从胎盘表面的蜕膜组织（白灰色层）。执行此步骤仔细维持胎盘及蜕膜组织（ 图1G）的完整性。 </li><li>放置在两个独立标记的培养皿填充用1×PBS溶液中的胎盘和蜕膜（3 - 各5ml）。 </li><li>轻轻地从尿囊膜用细尖镊子取出子宫壁。这些是子宫组织。 </li><li>将子宫组织在培养皿中填充用1×PBS溶液（3 - - 5毫升）中。 </li><li>重复步骤1.8至1.15，直到所有植入部位都已经被处理。 </li><li>收集几个子宫，蜕膜和胎盘组织，以改善细胞的酶消化过程中的产率。组织的量取决于产仔;然而，白细胞隔离要求&gt; 2个组织。 注:有关在不同妊娠天着床部位的解剖更详细的信息，查看的指南Investigati第二章中的图片对小鼠妊娠42。 </li></ol> 2.子宫，蜕膜和胎盘组织离解<ol><li>标号几个无菌2毫升锥形管具有适当的组织名称，并放置一个小瓶1000微升在冰上酶溶液。 </li><li>从培养皿填充用1×PBS溶液，转移两块 - 子宫，蜕膜或胎盘组织的（100 150毫克）到一个标记为2毫升锥形管中。 </li><li>加入500微升冷酶溶液成每次2毫升锥形管中。 </li><li>用细无菌剪刀，开始分解浸泡在酶溶液的组织，直到它被剁碎。随着时间的推移，暂停将开发一个乳白色的外观。此步骤不应超过超过2分钟，以防止过度操纵样品。 </li><li>一旦组织已被解离，添加一个额外的500微升冷酶溶液以2毫升锥形管中。 </li><li>放置2毫升锥形管在冰上。 </li><li>重复步骤2.2至2.6与每个单独的组织，直到所有的组织都已经被处理。 </li><li>转让全部2毫升锥形管与组织匀浆（组织糜+酶溶液）的样品从冰变成一个孵化器在37ºC和执行温和的轨道搅拌（80转）。温育30 - 35分钟。确保培养箱的温度开始培养时间之前是稳定的。 </li><li>孵育后，除去2毫升锥形管中从培养箱解离组织，并将其放置在冰上，以防止任何进一步的消化。 </li><li>按照组织类型的标签无菌50ml锥形管。取下盖子，并将它们与一个无菌细胞过滤器（100微米孔径）替换。 </li><li>倾从2毫升通过细胞过滤锥形管使用离解的组织约20毫升的1×PBS溶液。组织的碎片会保留在细胞过滤并将细胞悬浮液将通过它。 </li><li>洗迪在细胞过滤ssociated组织2 - 3次用20ml的1×PBS溶液，用移液管。 </li><li>冲洗空2毫升锥形管用1×PBS溶液（1毫升）中，并通过细胞过滤器倾的内容。 </li><li>从取出50毫升锥形管的细胞过滤器和更换盖子。离心管在1250×g离心10分钟，在4ºC。 </li><li>仔细，吸出上清液，而不会干扰细胞沉淀。将细胞沉淀包含所述分离的白细胞和基质细胞。 </li><li>重悬在1ml的RPMI培养基中的细胞沉淀无胎牛血清（FBS）。轻轻混匀。不要使用旋涡。 </li><li>加入500μl整齐的FBS为5ml的聚苯乙烯塑料管，慢慢地覆盖细胞悬液。 </li><li>离心机在1100×g离心10分钟，不制动在RT以沉淀的活细胞，而细胞碎片被保持在PBS / FBS的接口。 </li><li>吸出上清液而不触及细胞沉淀。该CEL升颗粒主要含有活细胞。 </li><li> 任选:要集中的单核细胞，用20％的饱和多糖溶液，如下所述。 <ol><li>重悬在1000微升的RPMI培养基中的细胞沉淀无FBS中。 </li><li>加入1毫升20％饱和多糖溶液以5ml的聚苯乙烯塑料管和慢慢重叠在该溶液顶部的细胞悬浮液，根据制造商的说明。而层叠的样品，避免混合与细胞悬浮液在20％的饱和多糖溶液。 </li><li>离心机摆动出转子在500 XG 30分钟，在室温下不带刹车。它是关闭的制动，以避免混合细胞和丢失的梯度非常重要。单核细胞将在20％的饱和多糖溶液和1×PBS溶液之间的界面上被发现。 </li><li>吸并弃去上清液。将细胞沉淀主要含有单核细胞。 没有TE:要执行的可行性染色和细胞内染色，见步骤3.要进行免疫，请参阅步骤4.要进行可行性染色，只有外染色，见第5步要执行分离白细胞的细胞培养，见步骤6.要执行磁性细胞分选，见第7步。 </li></ol></li></ol> 3.活力染色的细胞可固定<ol><li>重悬在1100微升的1×PBS溶液中的细胞沉淀。轻轻混匀用微量移液器。 </li><li>转移100微升细胞悬浮液到5ml的聚苯乙烯塑料管。加入400微升的1×PBS溶液向5毫升聚苯乙烯管中。该管是用于组织自发荧光的控制。储存在4ºC，直到步骤3.5。 </li><li>传输剩余1000微升细胞悬浮液到一个新的5毫升聚苯乙烯塑料管。加入1μl的生存能力染料并轻轻均质。孵育在黑暗中30分钟，在4ºC。 </li><li>孵育后，添加1000＆＃181;升1X PBS液。用手轻轻混匀。 </li><li>离心机既5毫升聚苯乙烯塑料试管（步骤3.2和3.3）以1250×g离心10分钟，在4ºC。吸并弃去上清液。 注意:将细胞沉淀（步骤3.3）将用于在第4步将细胞沉淀（步骤3.2）将作为组织自发荧光的控制。 </li></ol> 4.免疫分型<ol><li>重悬细胞沉淀中加入50μl抗小鼠CD16 / CD32的（稀释1:在FACS缓冲液100 [0.1％BSA，0.05％叠氮化钠，1×PBS溶液，pH为7.4]）。轻轻混合细胞悬液。不要使用漩涡。 </li><li>孵育细胞悬浮液中在黑暗中的5毫升聚苯乙烯管中，在4℃下进行10分钟。 </li><li>孵育后，加入50微升每种抗白细胞单克隆抗体与细胞外的细胞表面标记或同种型匹配对照抗体反应（抗体稀释液必须验证）。例如，为了分析白细胞亚群，使用防摩使用的抗体与下列白细胞的细胞表面标记物进行反应:CD45，Ly6G，F4 / 80，的CD11c，CD49b，B220，CD3，CD4，和CD8（ 表1）。 </li><li>一旦抗外标记已被添加到中的5毫升的聚苯乙烯塑料管，轻轻混匀。孵育在黑暗中为30分钟，在4℃。 </li><li>在这段温育，准备和预温热的固定缓冲液（用去离子水稀释，1:5）如果需要的话，以37ºC，。保持在缓冲液中于37ºC在黑暗中直到它的使用是必要的。 </li><li>经过30分钟孵化，在中的5毫升的聚苯乙烯塑料管加入500μlFACS缓冲液，并轻轻拌匀。此步骤是必要的，以洗涤细胞，并除去过量的未结合的抗体。 </li><li>离心样品在1250×g离心在4℃下进行10分钟。吸并弃去上清液。 注意:如果不需要的细胞内染色，参见步骤5。如果细胞内染色是必需的，则执行透和小区内ular染色这里，然后进行固定（步骤4.8）。 </li><li>添加预热的固定缓冲液中的细胞沉淀在500微升并在37ºC孵育在黑暗中10分钟。 </li><li>免除500微升FACS缓冲成中的5毫升的聚苯乙烯塑料管。轻轻混匀。 </li><li>离心样品在1250×g离心在4℃下进行10分钟。吸并弃去上清液。 </li><li>加入500微升FACS缓冲液到每个样品中。这些样品现在可以通过流式细胞术进行分析。立即获取的样品，以取得最佳效果。 </li></ol> 5.活力染色的细胞无法修复<ol><li>胞免疫后，重悬在500μlFACS缓冲液将细胞沉淀。 </li><li>加入1微升4&#39;，6-二脒基-2-苯基吲哚二盐酸盐（DAPI，200微克/毫升）刚刚采集样品之前。 </li><li>使用流式细胞仪加入DAPI后2分钟 - 可视内1的样品。 </li></ol><ol><li>准备在无菌通风柜的工作站。使用水浴预暖的RPMI培养基至37℃。 </li><li>重悬细胞沉淀1000微升FACS缓冲液用于细胞计数。 </li><li>计数存活的细胞用自动细胞计数器或血球和0.4％台盼蓝溶液的数量，下面的制造商的说明。记录总活细胞计数。 </li><li>离心将细胞在1250×g离心在4℃下进行10分钟以去除FACS缓冲液。 </li><li>轻轻吹打2至3倍重悬在500μl预热的RPMI培养基中的细胞沉淀。 </li><li>板单元的期望数量在无菌的24孔板中并在37℃。孵化时间将所研究的问题而定。 </li></ol> 7.磁性细胞分选<ol><li>设置一个干净的工作站与下列物质:MS色谱柱，磁性细胞分离器，15毫升锥形管，30微米预分离过滤器，和一个多底座。 </li><li>重悬在500μl的MACS缓冲液（0.5％BSA，2mM的EDTA，1×PBS溶液，pH7.2）中的细胞沉淀。 </li><li>使用自动细胞计数或血球和0.4％台盼蓝溶液，计数和记录总活细胞计数。 </li><li>离心细胞在1250×g离心在4℃下进行10分钟。吸并弃去上清液。添加另一个500微升MACS缓冲。 </li><li>继续执行磁选按照制造商的建议。 </li><li>确定纯度通过流式细胞仪或显微镜。 </li></ol>

作者披露利益冲突。

它记录白细胞浸润的数量和表型特征在母胎界面一致的数据收集是必不可少的理解妊娠并发症的发病机制。几种技术已经描述了促进浸润从在母胎界面的鼠组织白细胞整个怀孕31,38,39,43-46的分离。然而，每种技术不同的是，使用不同的酶或酶组合，需要不同的解离时间，不指定组织的数量时，以及最重要的是，并不总是指定分离的细胞的生存能力。本文所描述的协议允许渗透从鼠组织白细...

免疫耐受怀孕是一段当母亲的免疫系统内发生截然不同的变化。这些变化让母亲忍受了胎儿，一个半同种异体移植物1。胎儿的父亲表示主要组织相容性复合体（MHC）抗原2，和胎儿细胞已经在母体循环3被发现;然而，胎儿不被拒绝4,5。这个谜尚不完全清楚。 最新的假说认为，母胎耐受性性交过程中创建和受精6,7和维护，以维持一个足月妊娠8-10。这母胎耐受的细目怀孕期间的10-16早期和晚期疾病视为一种机制。母胎容忍包括各种白细胞亚种群，包括T细胞（调节性T细胞，Th1细胞，Th2细胞，和Th17细胞），MAC的参与噬细胞，嗜中性粒细胞，肥大细胞，NK细胞和NKT细胞，树突细胞，和B细胞，在密度和本地化这种变化在整个孕期15,17-19。母胎耐受富集在母胎界面20 -解剖位置在哪里，母亲的免疫系统与胎儿抗原相互作用20,21。 母胎界面胎盘期间，当胎儿绒毛外滋养细胞侵入子宫粘膜22-24创建。在该接口的胎侧，围绕胎儿膜创建一个专门的上皮表面的胎盘内，并且合体细胞控制与母体血液22通过其直接接触的物质交换。在界面的母体侧，蜕膜募集在小鼠占30％至所有蜕膜细胞的50％的白细胞的异质池。除了它们在matern参与人免疫耐受，这些细胞是主要贡献者怀孕期间不同的过程， 例如 ，在保护免受感染，受精生殖道，胚胎着床7.25，蜕膜血管26，血管重构24,27，滋养细胞浸润28，胎盘发展24,25，以及最终，生产和分娩15,17。因此，参与母胎耐受性白细胞的研究是必不可少的阐明了妊娠并发症的发病机制。 虽然使用免疫组织化学和免疫已产生的数据的直接可视化和子宫，蜕膜，胎盘或白细胞29,30的定位，流式细胞仪分析进一步揭示白细胞的特定子集在这些组织中31,32。此外，流式细胞仪已被用来确定密度和脑膜的比例NAL-母胎界面白细胞33和表达水平外和细胞内的蛋白质8-10,34的。流白细胞术分析在母胎界面需要一个单细胞悬液。为了从蜕膜，子宫和胎盘组织中分离白细胞浸润，组织离解的两种方法已被用于:机械和酶。这两种方法都允许渗透白细胞从这些组织的细胞外基质（ECM）的分离。酶组织离解优于机械组织离解，因为它允许更高的产率白细胞较少剪切力相关损害35。因此，机械组织解离需要汇集组织36，其可以增加样品的变异性和非均质性。然而，机械解离可能是选择，当目的抗原可以通过酶解或当改变细胞的功能感兴趣的需要s到被保留（ 例如 ，NK细胞的细胞毒性）35。 使用蛋白水解与特定的酶降解的ECM消除了低收率和机械解离观察。一些研究报告用32胰蛋白酶，胶原酶37，DNA酶31，分散酶38，以及各种酵素32,39的商 ​​业鸡尾酒。然而，性质和不同的酶浓度和消化的持续时间，必须精心定义和验证，以确保维持所需的免疫细胞表面抗原性表位的完整性。各表面结构是由不同的酶差异易受破坏，与某些酶，如胰蛋白酶，是臭名昭著的汽提通过许多单克隆抗体所识别白细胞表面的表位。 这里介绍的是使用proteolyt的方法IC和胶原溶解酶的鸡尾酒，叫的Accutase。此酶溶液是温和足够，而在在母胎界面解离鼠组织仍然有效，并且不需要加入其它离解的试剂或血清以终止分解反应。而且，它已准备好使用所提供，虽然需要解离的时间进行验证，它比上面提到的酶40,41更加健壮。 这两种类型的组织分解的组合的利用率提高了获得的细胞的质量和数量;因此，一些研究已经实施了机械和酶解结合使用，获得满意结果31,32,37。本文所描述的协议建立和验证在我们的实验室;它使用了一个温和的机械解离，随后通过一个健壮的酶分解的组合。该协议允许的分离和进一步研究在白细胞浸润在小鼠组织在母胎界面（子宫，蜕膜，胎盘）。以下协议也保持了细胞表面标记物和产率足够的活细胞用于下游应用程序的完整性就证明了流式细胞术分析。最后，该协议维持细胞制备的分析和构成母胎界面不同小鼠组织的比较一致。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Magentic Cell Separation</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30&#956;m pre separation filters</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multistand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL safe lock conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer</td>
			<td></td>
			<td></td>
			<td>(0.5% bovine serum albumin, 2mM EDTA and 1X PBS)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse extracellular antibodies</td>
			<td></td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td>(BSA)</td>
		</tr>
		<tr>
			<td>LIVE/DEAD viability dye</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation buffer solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td></td>
			<td></td>
			<td>(1% bovine serum albumin, 0.5% sodium azide, and 1X PBS ph 7.2)</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</td>
		</tr>
		<tr>
			<td>Incubator with shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from the murine tissues at the maternal-fetal interface

免疫耐受妊娠需要母亲的免疫系统发生，以接受和培养胎儿发育截然不同的变化。性交过程中启动了这种宽容，受精和着床过程中建立和维护整个孕期。母胎耐受性活跃的细胞和分子介质富集在胎儿与母体组织，被称为母胎界面，其中包括胎盘与子宫及蜕膜组织之间的接触部位。这个接口是由间质细胞和浸润白细胞，和它们的丰度和表型特征在怀孕过程中发生变化。白细胞浸润在母胎界面包括嗜中性粒细胞，巨噬细胞，树突细胞，肥大细胞，T细胞，B细胞，NK细胞，和NKT细胞一起创建的局部微环境，维持妊娠。这些细胞或任何inapprop之间的不平衡riate改变其表型被认为是疾病的妊娠的机制。因此，渗透到母胎界面白细胞的研究，以阐明导致妊娠并发症的免疫机制至关重要。本文中所描述的是，采用温和机械解离，随后通过一个健壮酶促解聚用蛋白分解和溶胶原酶鸡尾酒隔离从鼠组织中的白细胞浸润在母胎界面的组合的协议。该协议允许对高数量可行白细胞（&gt; 70％）以足够保守抗原性和功能特性的分离。孤立白细胞然后可通过多种技术，包括免疫，细胞分选，成像，免疫印迹，mRNA的表达，细胞培养，并在体外功能测定法如混合白细胞反应，增殖或细胞毒性测定法来分析。

从母胎界面小鼠组织的解剖示于图1;此过程包括打开腹膜腔（ 图1A，B），子宫角（ 图1C），包括植入位置（ 图1D），和子宫组织的集合（ 图1E），胎盘（ 图1F）和蜕膜（ 图1G），在16.5 DPC。 图2示出了分离的巨噬细胞的形态（F4 / 80 +）的蜕膜和子宫组织在16.5 DPC使用磁性细胞分选收集的。孤立的巨...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface at JoVE.com

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

isolation-leukocytes-from-murine-tissues-at-maternal-fetal

Research

JoVE Journal

Immunology and Infection

17.1K 次观看.  Wayne State University School of Medicine. Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis. 

视频: Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发:协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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 ...