这项工作是由儿童健康和人类发展的尤尼斯·肯尼迪·施莱佛国立研究所，美国国立卫生研究院/ DHHS支持。这项工作也得到了支持，在某种程度上，在孕产妇，围产期和儿童健康的韦恩州立大学围产期倡议。 我们非常感谢莫林McGerty（韦恩州立大学）为她的手稿的读数。

该协议适用于由底蜕膜及蜕膜在顶叶准备免疫流式细胞仪检测白细胞隔离。此外，可用于细胞分选，细胞培养，RNA分离和细胞学分离的细胞。与之前在此协议中提到的样品的工作，人类伦理委员会批准，必须从本地研究伦理委员会和伦理审查委员会获得的。收集和利用人力样本用于研究目的的批准了儿童健康和人类发展（NICHD）的尤尼斯·肯尼迪·施莱佛国立研究所的机构审查委员会，健康（NIH）全国学院，健康与人类服务部（DHHS ，贝塞斯达，MD，USA）和韦恩州立大学（底特律，MI，USA）。书面知情同意从所有孕妇之前组织样本的采集获得。 注意:当有动物血，细胞，或危险工作代理的OU中提到这个协议中，很重要的是正确的生物安全和实验室安全行动应遵循。 1.解剖人类蜕膜的注:基板是下方并附着到胎盘底座和表示母体的表面。该chorioamniotic膜包括羊膜和绒毛膜。基底板包括蜕膜和绒毛膜包括蜕膜顶叶（ 图1）。 <ol><li>底蜕膜<ol><li>从胎盘（ 图1A和1B）中的一个子叶解剖一块基底板。 </li><li>将其放在无菌砧板与朝上胎盘绒毛。侧面显示了胎盘绒毛通常是血淋淋的红色外观毛茸茸的组织。基底板是平滑和颜色淡红色。 </li><li>用锋利，细点的剪刀和镊子除去VILLOU中组织和血管。保持浸泡在1×PBS中的组织的过程中（ 图1C）中（ 钙 -自由-和Mg 2+）。收集2-3这些片和用1×PBS彻底冲洗以除去血液（ 图1D）。 </li></ol></li><li>蜕膜顶叶<ol><li>解剖一块chorioamniotic膜（约10厘米×10厘米， 图1E）。将它放置在黑板上用绒毛膜朝上。绒毛膜侧包含血腥和血块通常是淡黄色的颜色。 </li><li>用细点钳去除尽可能多的血块为可能的（ 图1E）。冲洗该膜在无菌的1×PBS清洗过量的血液从该膜，直至1×PBS中运行清楚。 </li><li>使用一次性无菌细胞刮至轻轻刮去从膜的蜕膜层。应用无菌1X PBS的纪念品brane而完成这个过程（ 图1F）。收集蜕膜组织，并放置在50毫升的塑料管，用无菌1X PBS（ 图1G）。 </li></ol></li></ol> 2.机械解聚和酶消化<ol><li>在50ml塑料管中洗，用无菌的1×PBS从蜕膜（来自步骤1.1.3）解剖组织或蜕膜顶叶（来自步骤1.2.3）。通过离心收集组织粒料在300×g离心5分钟，在RT。 </li><li>吸位于高于组织沉淀上清小心而不会干扰沉淀。 注意:在这一点上，该颗粒是非常宽松，因为它们包含红血球。如果粒料的体积为约或低于3毫升，再悬浮在6ml的细胞脱附溶液预加热至37℃沉淀。如果粒料比3毫升体积较大，增加更多的细胞脱离溶液（日增加约2倍Ë体积组织样本）。 </li><li>转移组织匀浆（底蜕膜+细胞分离液，或蜕膜+顶叶细胞分离液）为C管。 </li><li>代替C-管在自动离解器并运行相应的程序。 注:该程序为底蜕膜运行17秒，共668发每运行。该程序为蜕膜运行顶叶为37秒，共235发每运行。这些方案已自定义为从底蜕膜或蜕膜与顶叶良好的收益率和细胞活力隔离白细胞。 </li><li>以下的组织的机械解聚，消化组织中的市售的细胞分离溶液如的Accutase;为45分钟，在37℃，轻轻摇动（包含蛋白水解和胶原溶解酶鸡尾酒）。 </li></ol> 3.隔离白细胞<ol><li>孵育后，加入10 mL的1×PBS中的DIGEStion的混合物中，并通过100微米的细胞过滤将它传递到50ml塑料管中。取决于消化混合物的粘性，人们可能需要用几个细胞滤器一混合物。 </li><li>填充管用1×PBS和离心机在300×g离心5分钟，在RT。取出的1×PBS上述细胞沉淀小心和重新悬浮用5ml冰冷却的FACS缓冲液中的颗粒。 注:要研究多形和单核白细胞在一起，则转到步骤6.1;研究单核白细胞，继续执行步骤3.3。 </li><li>加入5毫升20％的密度梯度介质（1.077 + 0.001g /每毫升），以15毫升的塑料管中并慢慢叠加在密度梯度介质的顶部的细胞悬浮液。而层叠的样品，它不与细胞悬浮液混合的密度梯度介质是重要的。 </li><li>离心用水平转子在500×g离心30分钟，在4℃不制动。关闭制动器，以避免混合细胞和L这是非常重要的osing梯度。白细胞将在密度梯度介质和FACS缓冲液之间的界面上找到。 </li><li>删除包含FACS缓冲液和细胞碎片非常仔细地用移液管上层。界面处的频带包含蜕膜单核细胞和多形核白细胞的一部分。 </li><li>完全转移的细胞在界面到一个新的15毫升塑料管。加的FACS缓冲液的至少3倍以上的体积。 </li><li>离心在300×g离心5分钟以造粒细胞。吸出上清液，并用FACS缓冲液洗细胞。造粒的细胞再次离心分离，在300×g离心5分钟，在RT。 注:要执行的细胞培养，见第4步:为了进行细胞分选，见第5步:为了进行免疫，见第6步。 </li></ol> 4.应用 - 细胞培养<ol><li>重悬在1ml的RPMI培养基1640的来自步骤3.7的细胞计数的存活细胞USI纳克自动细胞计数或血球和台盼蓝溶液0.4％。 </li><li>加起来的RPMI培养基的量，使1×10 6个细胞/ ml的细胞浓度。细胞现在已经准备好了的文化。 </li></ol> 5.应用程序 - 巨噬细胞分离的原代细胞培养<ol><li>以分离的CD14 +细胞，磁来自步骤3.7与CD14微珠（每10 7个细胞20微升磁珠）标记细胞，孵育15分钟，在4℃，通过施加磁场单独的CD14 +细胞从其它类型的细胞。洗涤2次后用MACS缓冲液和除去磁场，洗脱磁力保留CD14 +细胞用MACS缓冲液。 </li><li>离心后，重新悬浮细胞沉淀在RPMI培养基1640中细胞准备用于电镀（ 图2）。 </li></ol> 6.应用 - 免疫分型<ol><li>以使用该单元为immunophenotyping，在1ml的1×PBS再中止来自步骤3.7的细胞。计数使用自动细胞计数器或血球和台盼蓝溶液0.4％的活细胞。 </li><li>标记的细胞与可固定活力染料（ 图3）。用FACS缓冲液清洗细胞两次，并重新悬浮细胞在染色缓冲液中。孵育一个的FcR阻断剂为15分钟，在4℃。 </li><li>添加共轭荧光抗体外。例如，在这个协议中，使用抗CD3，抗CD19，抗CD14，抗CD15和抗CD56抗体（ 表1）。温育30分钟，在黑暗中4℃。 </li><li>洗涤2次后用1×PBS，细胞在500μl的染色缓冲液使用流式细胞仪进行分析。用于分析在蜕膜组织中发现的白细胞亚种群的门控策略的表示示于图4。 </li></ol>

The authors disclose no conflicts of interest.

在人类母胎界面白细胞浸润的功能和表型特征的表征是必不可少的，导致妊娠疾病的免疫机制的理解。若干技术以分离从人母胎界面白细胞整个怀孕10,14,25,28,37,42,43了描述。然而，每一个这些技术是截然不同的，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力。本文所描述的协议允许浸润在人类母胎界面白细胞（蜕...

怀孕的特征在于三种不同的免疫学阶段:1）植入并用促炎症反应相关的早期胎盘（ 即 ，植入类似于一个"开放性伤口"）; 2）孕中期和孕大部分的孕晚期时免疫稳态通过主要是消炎的状态在母胎界面实现; 3）分娩，促炎症状态1-7。免疫细胞在母胎界面发挥在炎症反应的调节中起重要作用，他们的丰度和在整个孕期6-9本土化的改变。 在人类中，母胎界面代表了母性（蜕膜）和胎儿（绒毛膜滋养层或）组织之间的直接接触面积。该接口包括:1）蜕膜顶叶的行宫腔未包括的胎盘和是并列以绒毛膜绒毛的;和2）的底蜕膜，位于它是由间质滋养层10（ 图1）侵入胎盘的基板。接触的这些区域的亲密创建胎儿抗原暴露于母体免疫系统11-13的条件。不足为奇的是，白细胞包括多达蜕膜细胞8,9,14,15 30-40％除了典型的基质型细胞和腺细胞8,14,16。白细胞在母胎界面的作用涵盖了包括滋养细胞浸润17的限制，螺旋动脉18,19的重塑，维护产妇宽容12,20和劳动力21-26启动多个进程。两者的适应性和免疫系统， 即 ，T细胞，巨噬细胞，中性粒细胞，B细胞，树突细胞，和NK细胞，已在蜕膜组织确定了先天四肢，以及它们的白细胞比例和激活状态已示出，以改变空间和时间整个妊娠期6-10,12,14,24,27-30。在白细胞种群和/或功能的扰动与自然流产31，先兆子痫32，宫内生长受限32,33，和早产7,24相关联。因此，在人类母胎界面的表型特征和白细胞功能的研究将有助于失调在怀孕紊乱的免疫途径的阐明。 一种用于确定表型和白细胞的功能特性被流式细胞术的最有力的工具，技术，允许多个参数同时34-36的定量分析。分析白细胞通过流式细胞术，在单细胞悬浮液中的白细胞的隔离是必需的。因此，一个方法来分离浸润亮氨酸从母胎界面kocytes是需要研究其表型和功能特性。 几种方法已经被描述，从人类母胎界面10,14,25,27,37-39隔离白细胞。虽然一些应用机械分解10,25,27,38，别人用酶消化37,40进行组织分离。因为机械解聚产生较低产量和降低的生存力41，和酶解可影响生存力和细胞表面标记物保留42，本文所描述的方法结合温和机械解离与酶预处理，以增加分离的白细胞的产量而不影响细胞生存力。方法类似的组合已经被证明是有效的，从在母胎界面39蜕膜组织白细胞的隔离。因此，本文描述的协议涉及机甲nical分解与自动组织离解，增加一致性，同时节省时间和劳动时与反对手术刀，刀片，或手术剪10,28比传统的切碎。选择用于组织解离酶是的Accutase。不同于常用的胶原酶43，分散酶44，和胰蛋白酶45的Accutase（细胞分离液）结合了一般的蛋白和有助于高效而温和的解离46,47胶原溶解活动。解离后，将白细胞从蜕膜细胞通过密度梯度离心的总人口富集。各种密度梯度介质先前已经利用，其中最常见的是珀（胶体二氧化硅粒子的悬浮液）48和聚蔗糖（蔗糖的具有高合成分子量的聚合物）49。隔离的蔗糖聚合物的卓越效率一直previously所示50，和本文中进一步描述的方案证明该密度梯度介质能产生单核白细胞的足够高的纯度。 因此，本文描述的协议相结合的机械组织分解用自动组织离解，酶消化用细胞分离溶液，并用密度梯度介质（1.077 + 0.001g /每毫升）以分离从人蜕膜白细胞白细胞的分离。该协议已经被证明维持细胞表面抗原连同细胞活力。分离的白细胞可用于多种应用，包括免疫流式细胞仪和体外功能研究。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Sterile dissection tools: surgical scissors, forceps, and fine-tip tweezers&#160;</td>
			<td>Any vendor</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td></td>
			<td>(1X PBS)</td>
		</tr>
		<tr>
			<td>Large and small Petri dishes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissociation</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td>(cell detachment solution)</td>
		</tr>
		<tr>
			<td>Sterile 2 mL safe-lock conical tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainers</td>
			<td>FALCON/Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom polystyrene test tubes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>RPMI culture medium 1640&#160;</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td>(1X) (10% FBS and 1% P/S)</td>
		</tr>
		<tr>
			<td>Plastic chamber slides</td>
			<td>Thermo Scientific</td>
			<td>177437</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific Corporation</td>
			<td>HEPA Class 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>ISOTEMP 110</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Nexelcom</td>
			<td>Cellometer Auto2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Olympus CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Separation</td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#956;m pre-separation filters</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL safe-lock conical tubes</td>
			<td>Any Vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS buffer&#160;</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>FcR Blocking</td>
			<td>Miltenyi Biotec</td>
			<td>130-059-901</td>
			<td>(Fc Block)</td>
		</tr>
		<tr>
			<td>Anti-human cell surface antigen antibodies&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Bovine serum albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD viability dye</td>
			<td>BD Biosciences</td>
			<td>564406</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyse/Fix buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>346202</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td></td>
			<td></td>
			<td>(1% BSA, 0.5% Sodium Azide, and 1X PBS)</td>
		</tr>
		<tr>
			<td>Staining buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>20% density gradient media</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</td>
		</tr>
		<tr>
			<td>Incubator with shaker</td>
			<td>Thermo Scientific</td>
			<td>MAXQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSR-Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>SpinChron DLX</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum system</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic tissue dissociator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>gentleMACS Dissociator</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from the human maternal-fetal interface

怀孕的特点是在生殖组织，并在母胎界面白细胞（底蜕膜及蜕膜顶叶）的渗透。此接口是母体和胎儿组织之间的接触解剖位置;因此，它是作用在怀孕期间免疫位点。白细胞浸润在母胎界面起到交付的植入了核心作用，维护妊娠和时序。因此，这些白细胞的表型和功能性的刻画将提供洞察导致怀孕紊乱的机制。有几个协议，以隔离从蜕膜和蜕膜顶叶白细胞浸润了说明;然而，由于缺乏在试剂，酶和孵化的时间一致性使得难以比较这些结果。本文描述的是一种新的方法，结合使用温和的机械和酶迪斯引产技术，以保护细胞外和细胞内标志物的可行性和完整性白细胞从在母胎界面的人体组织中分离。除了免疫，细胞培养和细胞分选，这个协议的未来的应用是多种多样的。按照此协议，分离的白细胞可用于确定DNA甲基化，靶基因的表达， 在体外白细胞功能（ 即 ，吞噬作用，细胞毒性，T细胞增殖，和可塑性， 等 ），以及生产的活性氧在母胎界面。此外，使用所描述的协议，该实验室已经能够描述新和罕见的白细胞在母胎界面。

。在母胎界面人类组织（底蜕膜及蜕膜顶叶）的解剖见图1此过程包括基板，其中包括底蜕膜（ 图1A - D）的清扫。通过除去从基板（图1C）的胎盘绒毛（胎侧）得到的蜕膜。蜕膜是顶叶轻轻刮绒毛膜（ 图1E - F）收集。 图2显示了孤立的巨噬细胞（CD14 +），从使用磁性细胞分选在足月妊娠蜕膜收集顶叶的形态。...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Human Maternal-fetal Interface at JoVE.com

Isolation of Leukocytes from the Human 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-leukocytes-from-the-human-maternal-fetal-interface

Research

JoVE Journal

Immunology and Infection

15.9K 次观看.  NICHD/NIH/DHHS. 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. 

视频: Isolation of Leukocytes from the Human 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

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. ...

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 ...

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

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

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 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 ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...