Name: characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols
Uploaded: 2016-10-18T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

总体人体血液的研究已经审查和金融情报机构，IRB协议批准＃IRB-13-0440的机构审查委员会（IRB）的批准。人类leukopaks从迈阿密，佛罗里达州社区血库购买。 1.外周血单个核细胞分离通过标准密度梯度法<ol><li>在T75烧瓶中的血液用1×磷酸盐缓冲盐水1稀释:执行1。 </li><li>移液器的15ml密度梯度溶液倒入50ml离心管中，小心地层（25 - 为30 mL /管）稀释血液在这个梯度。 </li><li>在1200 xg离心以1加速和0减速离心20分钟。 </li><li>离心后，收集界面层（白血细胞）到一个新的50ml离心管中并在PBS中洗涤细胞两次（1,080 xg离心3分钟）。丢弃每次上清。 </li><li>治疗与氯化铵 - 钾裂解（ACK）缓冲液细胞（裂解红血细胞）。加10毫升缓冲液中，cubate在4℃下15分钟。 </li><li> 1,080 xg离心洗涤细胞两次，用PBS，离心3分钟，弃每次上清液。 </li><li>保存颗粒，其中将包含外周血单个核细胞。 </li><li>与选择的单核细胞纯化方法进行。 </li></ol> 2.贴壁法提纯单核细胞<ol><li>制备含有L-谷氨酰胺（300毫克/毫升）和完整的细胞培养基补充有青霉素（50U / ml）的-streptomycin（50微克/毫升）和10％胎牛血清。 </li><li>允许的PBMC通过在T75烧瓶中以每10 ml完全培养基的5×10 7个细胞的浓度在37℃和5％CO 2在培养箱中培养他们坚持约2小时。 </li><li>培养后，从培养瓶中除去非贴壁悬浮细胞和轻轻地用PBS洗涤附着的细胞两次。 </li><li>孵育贴壁细胞在补充有2微升/ ml人粒细胞 - MACR的完全细胞培养基ophage细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）存储在库存浓度为10微克/毫升。 </li><li>改变介质的一半，并补充细胞因子每48小时。 </li><li>允许5 - 7天为单核细胞分化成MDDCs。 </li></ol> 3.单核细胞通过磁分离方法来纯化<ol><li>收集从标准密度梯度技术的PBMC，倒入5毫升聚苯乙烯管中并在PBS缓冲液中的细胞/ ml的浓度重新悬浮。 </li><li>加入人单核细胞富集的鸡尾酒用50微升/毫升细胞，拌匀，并在4℃孵育10分钟。 </li><li>孵育后，加用50微升/毫升细胞的磁性颗粒，拌匀，并在4℃孵育5分钟。 </li><li>通过加入PBS缓冲液使细胞悬浮液至2.5毫升总体积，通过上下吹打2拌匀 - 3次。 </li><li>放置聚苯乙烯管无帽到磁性装置，并在室温进行2预留0.5分钟。 </li><li>温育后，在一个连续的动作，拿起磁铁和倾所需纯化的单核细胞级分成50 mL的干净的离心管中。 </li><li>孵育在补充有2微升/ ml人粒细胞 - 巨噬细胞集落刺激因子（GM-CSF）的完全细胞培养基和白介素4贴壁细胞（IL-4）存储在库存浓度为10微克/毫升。 </li><li>每48小时，更改一半培养基，降速1,080×g离心5分钟，重新悬浮新媒体（CRPMI）和细胞因子粒料。 </li><li>允许5 - 7天为单核细胞分化成MDDCs。 </li></ol> 4.台盼蓝染色活性测定 注意:使用此技术来获得外周血单个核细胞，单核细胞和MDDCs的细胞产量和活力。 <ol><li>收获细胞用标准胰蛋白酶-EDTA，并执行烧瓶并用PBS将细胞沉淀的洗涤。根据粒料的大小，重暂停在5至10毫升的PBS以溶解沉淀。 </li><li>在微型离心管中，执行1:锥虫蓝试剂用稀细胞沉淀1稀释。 </li><li>从这种混合，分装10微升到细胞计数滑动和使用根据制造商的协议的自动细胞计数器读取的结果。如果自动细胞计数器不可用，类似的细胞计数可以使用血球或手动计数器。 </li></ol> 5. MTT法<ol><li>板细胞的浓度为4×104 /100μl的完全培养基的每孔在96孔板中。允许细胞调整到培养24小时。 </li><li>孵育并在分别为0（0天），36（第3天）和84（第7天）小时进行读数。 </li><li>在所需的孵化完成后，取出媒体和单独100微升的PBS替换。 </li><li>制备3-（4,5-二甲基吡啶-2-基）-2,5-二苯基四唑鎓溴化物溶液（MTT）（5毫克/毫升）中加入10毫升dimeth的基砜（DMSO）至1g十二烷基硫酸钠（SDS）。 </li><li>添加10微升（5毫克/毫升）的MTT溶液，以每孔，并在37°C孵育另外2小时。 </li><li>孵化后，取出含有PBS和未MTT混合物（黄色溶液）上清。 </li><li>加入100微升的SDS-DMSO溶液至每孔，并孵育一小时。 </li><li>在使用酶标仪540nm处读取吸光度。 </li></ol> 6.细胞表面染色分析成像流式细胞仪<ol><li>之后，从纯化的单核细胞7天分化，分配所述单核细胞来源的树突状细胞的1×10 6细胞的等分试样到1.5毫升管的适当数量。 </li><li>通过使用热50μl的灭活（HI）的人血清阻断细胞开始细胞表面染色，孵育在4℃下进行10分钟。 </li><li>培养后，离心细胞以720×g离心5分钟，弃上清。 </li><li>细胞PELLET，添加各自荧光染料标记的抗体（ 例如，抗CD11c或抗CD14），并在4℃下孵育避光20分钟。在单独的管，制备单荧光染色样品/毫升），因为它们都需要赔偿。 </li><li>温育后，洗涤细胞两次，每次1ml的PBS缓冲液和离心机在720×g离心5分钟。 </li><li>保持避光细胞，在PBS缓冲液的细胞/ 100微升流式细胞仪分析浓度再挂起。 </li><li>为了对活细胞栅极，获取对单细胞成像流式细胞仪根据制造商的指示仪表的细胞之前，添加1微升的DAPI到每个管中。 </li></ol> 7.统计分析<ol><li>输入电子表格中的所有数据。 </li><li>比较使用学生t检验和/或单向ANOVA适当的结果。 </li><li>考虑差别是如果p &lt;0.05统计学显著。 </LI&gt; </ol>

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All authors disclose no competing financial interests.

根据分离和产生从人血MDDCs的已知困难，本研究的目的是提供用于MDDCs的产生两个成熟的方法的全面的比较。相比第一种方法是用于通过利用单核细胞粘附到玻璃或塑料（附着法）21,22,27的能力产生MDDCs行之有效的传统方法。的粘附方法是快速和成本有效的，并且不要求使用复杂的设备。然而，这种方法的一些缺点包括淋巴细胞污染，低弹性，单核细胞瞬时操纵22,30,31。相比，第二方案的方法是使用一个人单核细胞富集试剂盒26，其目的是通过阴性选择来隔离的PBMC的单核细胞总外周血单核细胞（PBMC）的单核细胞的磁隔离。这种分离方法的优点是，不需要的细胞标记，并使用一个磁铁分离而靶细胞可自由倾出进新管中，而不需要的列和不与抗体直接靶向细胞。无论单核细胞的分离方法的（附着的与磁分离），这两个程序后，的协议中的关键步骤之一是与粒细胞巨噬细胞集落刺激因子的单核细胞的体外刺激（GM-CSF）和白细胞介素-4（ IL-4），它被要求产生MDDCs。这是一种常见的和很好地建立协议，以产生从血液单核细胞MDDCs而不损害抗原捕获和未成熟MDDCs 32的处理能力的特性。一个常用的体外生成DC的技术的主要限制是前体细胞，如单核细胞的产率低。正如上面的报道，贴壁法分离出单核细胞占P =约6.2％骨髓细胞，而通过磁分离分离的单核细胞占的PBMC的高达25％。据文献报道，范围为正常成人微分白血球计数的单核细胞是从2 - 10％31而树突只占0.1 -总血单核细胞20的1％。因此，后磁分离获得的单核细胞的高收率（〜25％）可能是由于细胞群体和缺乏抗体和/或磁性粒子结合不需要的细胞的的杂质。 为了达到分离的单核细胞以高收率和纯度，所有在该协议的关键步骤，应密切注意。在从血液通过标准密度梯度离心的PBMC的分离，有几个关键步骤。首先，移液和过密度梯度液仔细分层的血液到50ml离心管中是需要吸取以来严厉可能会导致此解决方案和血液的混合实践的一个步骤，这会导致弱的PBMC层是很难分离，或者其可以导致红血细胞的PBMC的完全混合物。在此步骤中，它保持移液的恒定和相对缓慢的流动具有定义良好的血液层和一个不同的PBMC层是重要的。第二，离心与加速度1和减速0，下一步是非常关键的。如果未用于第一离心步骤这些设定，它会导致不规律的密度梯度溶液中的血的混合，并且不会对PBMC中从血液成分的其余部分分开。第三，孵育用氯化铵-钾（ACK）裂解缓冲液中的PBMC为不低于优化时间长是很重要的（10 - 15分钟），因为有ACK潜伏期较长可导致减少可行的PBMC与红细胞沿。此外，同样重要的是，进行ACK孵育后的PBS洗涤。然而，如果红细胞不与第一裂解ACK一步，他们仍然继续洗涤后出现，强烈建议新一轮ACK裂解的。 白血细胞分离后，有几个关键的步骤执行遵守方法来产生单核细胞时，要牢记。第一，二小时的PBMC培养后洗去浮动淋巴细胞是非常重要的，因为浮淋巴细胞的存在可导致更少的单核细胞粘附并且还可以导致较低的MDDC纯度。虽然这些洗涤步骤是关键的，过度的和苛刻的洗涤（超过建议， 即 ，两次）可导致洗掉粘附单核太大，导致低的单核细胞的产率。第二，孵育用完全培养基和细胞因子的细胞，因为细胞因子是负责的单核细胞分化成MDDCs是至关重要的。此外， 改变媒体和补充细胞因子的每48小时为细胞的分化和存活的重要。而变化的媒体，它也是重要的，以节省浮动MDDCs并返回它们放回培养用新鲜培养基和细胞因子一起，因为有一些MDDCs能够分化过程中从表面脱离。它也是重要的，以确保所述单核细胞是在推荐的浓度接种，附着和紧密间隔的，因为它们以生长需要细胞与细胞的接触。在进行磁选法生成的单核细胞时，有为了获得良好的产率和单核细胞的高纯度的几个关键步骤。首先，它是保持由制造商推荐的细胞浓度是至关重要的。同样重要的是要放置在磁体时不打扰聚苯乙烯管中，因为它可能导致更低的产量和更低的纯度。如上述提出的，这种技术会导致相比时的粘附方法的显著更高的单核细胞的产率（ 图1），这可能与其他C的存在厄尔。此外，当细胞通过流式细胞术特征，磁性方法示出双阳性细胞（CD11C + / CD14 +）的比例更高，其可以与其它细胞的存在，由于MDDC的不同阶段的存在相关联，或者也可以是分化。 体外 MDDC生成无论产率和纯度的另一个重要方面是，以产生可行的和增殖MDDCs的能力。目前的研究提供了证据，证明这两种纯化方法诱导MDDCs（ 图3）的增殖过程中培养7天所示通过在细胞代谢活性的增加。另外，从通过磁分离纯化的单核细胞MDDCs的产生表明增殖的显著更高速率比较由粘附（ 图3）纯化的单核细胞产生MDDCs。这些研究结果都符合以前的文献表明使得G从CD34 +细胞的DC eneration是增殖过程中由于增生DC祖细胞在人体血液中27，33的存在。但是，也有争议的调查结果表明单核细胞也可以分化成树突而不增殖34,35的任何证据。这是有关指出，有很多关于体外代DCS的争议以及有关区议会是否从增殖前体或单核细胞分化产生的。例如， 在体外从贴壁外周血细胞生产的DC已经显示也富集，其能够暴露于GM-CSF的27后扩散并也有证据表明DC的产量在存在来源的祖细胞GM-CSF和IL-4的不能超越起始单核细胞33的数量进行扩展。总体而言，目前的研究表明在周一的增殖增加ocytic细胞的文化，在7日导致整个MDDCs人群&gt; 70％的CD11c +表型（坚持= 72％±11与磁= 79％±19）。这是有关指出，当进行进一步的表型分析，虽然没有显著，由磁分离法分离出的单核细胞导致MDDCs具有较高的双阳性表型（粘附= 49％±7的CD11c + / CD14 +与磁性= 73％ ±15的CD11c + / CD14 +），而通过粘接分离单核细胞产生具有更高的单阳性表型（粘附= 23％±7的CD11c + / CD14-与磁性= 6％±7的CD11c + / CD14-）MDDCs。具有较高的双阳性表型（表面CD11c + / CD14 +）MDDCs可能正在分化的早期阶段，同时具有较高的单个阳性表型MDDCs可能正在分化的后期阶段。 总之，这项研究提供的证据支持阴性选择磁选再修改的当通过坚持法分离的单核细胞相比，E要分离的单核细胞产生与单核细胞存活率没有显著差异的单核细胞的产量最高。反过来，七天后，通过磁分离中分离出的单核细胞分化成与显著更高的增殖能力和表达双阳性（的CD11c + / CD14 +）表型的细胞的较高量MDDCs而不影响MDDC生存能力。当比较这两种方法，研究结果已经证明这两种技术以同时产生，其能够在培养物中增殖（ 图3）和分化（ 图4）插入的CD11c + MDDCs七天后的自这两种方法，得到&gt; 70％的CD11c + MDDCs单核细胞的能力。然而，进一步的分析看成熟标记可能证明是有用的，充分表征功能MDDC人口。总之，这两种方法都对的CD11c + MDDCs和省的分离有利的替代品IDE用于研究应用适当的产率，甚至可能是为未来的临床应用是有用的。该实验室目前正致力于MDDCs的一代，研究酗酒和滥用先天免疫系统的其他物质的作用，尤其是在这些物质的修改MDDC表型和功能的能力。因此，目前的研究将提供MDDC表征的基线和增强与进一步的实验，以继续进行，以阐明在药物滥用的情况下介导单核细胞衍生的树突细胞的功能的分子机制的能力。

树突细胞（DC）是先天和适应性免疫系统的基本介质。它们的功能来诱导初次免疫应答，促进免疫记忆的发展。这些细胞对于抗原捕获，迁移和T细胞的刺激主要负责，因此，被称为1的DC .Manipulation可以在各种不同的研究领域，并且在临床上被用于治疗不同的专业抗原呈递细胞（APC）炎性疾病如HIV 6,7，癌症8，自身免疫性疾病9，和过敏性反应10。区议会也被用于药物滥用研究，以解决未知的机制和途径，如与酒精依赖11-14，药物依赖13,15，和HIV感染和滥用药物16-19的组合有关的。这些正在进行的研究和未来研究研究我ñ免疫学领域作出的体外代DCS的研究非常重要。然而，存在与从人血液中分离的DC，因为它们只构成0.1相关联的若干困难-总血单核细胞20的1％。 迄今为止，一些对的DC 在体外的产生的行之有效的方法是由单核细胞21,22的塑料或玻璃粘附，密度梯度离心23，特异性标记基于分离的诸如磁激活细胞分选22，荧光激活细胞分选24，用葡聚糖包被的磁性纳米颗粒25，并用完全自动化的负小区选择26高度纯化的单核细胞的快速分离CD14 +单核细胞的阳性筛选。但是，选择的最佳方法仍存在争议。因此，为了改善直流生成技术，几种方法已经被开发，其中这些细胞的纯度，可以大大增加从外周血单核细胞（PBMC）27分离纯化的CD34 +祖细胞和单核细胞的分化。作为现有所提到的，用于产生单核细胞来源的树突细胞（MDDCs）一种广泛使用的和流行的方法是探索的单核细胞粘附到玻璃或塑料（附着法）21,22,27的能力。的粘附方法是，不需要使用复杂的设备迅速和直接的方法。然而，这种方法的一些缺点包括淋巴细胞污染，低弹性，和单核细胞的瞬态操作28。到的粘附方法的另一种方法是从总的PBMC的单核细胞的磁隔离，特别是与使用人单核细胞富集的试剂盒，其被设计为通过阴性选择26来隔离的PBMC的单核细胞的。在此过程中，不想要的细胞被定位为与四聚体去除抗体复合物和葡聚糖 - 包被的磁性粒子。这种分离方法的优点是，不希望的标记的细胞使用磁体而靶细胞可以自由地倾出进新管中，而不需要的列隔开。迄今为止，与可标记独特的细胞群体特异性的单克隆抗体的情况中，磁性分离技术已成为不只是一个附加的方法，但对于罕见细胞在免疫学领域的隔离是必要的。例如，技术，诸如磁性细胞与市售的顺磁磁珠纳米颗粒分拣提供了便利的研究和临床应用22,29的新方法的发展。此外，最近的调查研究DC一代单核细胞，从坚持和MACS技术方法相比采用分离的单核细胞22,30 MACS都表现出较高的DC纯度和可行性。 目前的研究礼物使用商业人单核细胞富集试剂盒1）通过粘附的单核细胞的分离和2）单核细胞分离通过阴性选择:两种方法人DCs的一代从单核细胞从PBMC中分离之间的比较。本研究提供的证据表明，当与由粘附方法分离的单核细胞相比，阴性选择磁选步骤以分离单核细胞产生具有在单核细胞存活率没有显著差异的单核细胞的产率最高。反过来，七天后，通过磁分离中分离出的单核细胞分化成与显著更高的增殖能力和表达双阳性（的CD11c + / CD14 +）表型的细胞的较高量MDDCs而不影响MDDC生存能力。总体而言，目前的研究从上面引用的先前研究不同，因为它表明这两种技术以同时产生能够增殖和分化成的CD11c的单核细胞的能力+MDDCs（&gt; 70％）后，在培养7天不影响他们的生存能力。此外，目前的做法提供了流式细胞仪成像流量不同的CD11c / CD14 MDDCs人口的第一次鉴定。 综上所述，由于区议会扮演关于免疫学领域研究的焦点角色，不同的参数必须考虑到他们是如何得出的，并用什么方法将它们在体外分离培养时加以考虑。因此，本研究的目的是提供对单核隔离两种不同的方法，以及这些方法的差异影响单核细胞活力和最终产量影响树突状细胞活力，增殖和表型的见解。这些发现将大大有助于免疫学的领域，并且将提供的DC隔离，纯化和表征的详细协议。

<table><tbody><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-5442-03</td><td>Must be used at RT</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>ACK Lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>RoboSep buffer</td><td>StemCell</td><td>20104</td><td></td></tr><tr><td>EasySep Human monocyte enrichment kit</td><td>StemCell</td><td>19059</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Bio-Rad</td><td>145-0101</td><td></td></tr><tr><td>FITC-Dextran</td><td>Sigma Aldrich</td><td>FD4-100MG</td><td></td></tr><tr><td>Trypan blue stain (0.4%)</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Synergy 2 multi-mode reader</td><td>Biotek</td><td>7131000</td><td></td></tr><tr><td>XTT Sodium salt bioreagent (XTT)</td><td>Sigma Aldrich</td><td>X4626-100MG</td><td></td></tr><tr><td>Dimethyl sulfoxide bioreagent (DMS)</td><td>Sigma Aldrich</td><td>D8418-500ML</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide (MTT)</td><td>Sigma Aldrich</td><td>m5655-500MG</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Bio-Rad</td><td>161-0302</td><td></td></tr><tr><td>Phenazine Methosulfate (DMSO)</td><td>Sigma Aldrich</td><td>P9626-1G</td><td></td></tr><tr><td>Inactivated (HI) Human Serum</td><td>Chemicon</td><td>S1-100ML</td><td></td></tr><tr><td>Accuri C6 Flow Cytometer</td><td>BD Accuri</td><td>653119</td><td></td></tr><tr><td>FlowSight Amnis Flow Cytometer</td><td>EMD Millipore</td><td>100300</td><td></td></tr></tbody></table>

characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols

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

<html> 通过磁分离的单核细胞收率较高通过贴壁法相比单核细胞产量 在外周血单个核细胞和单核细胞的分离隔离当天由台盼蓝排除法图1显示PBMC和单核细胞计数显示的数据。平均来说，通过粘附方法分离的单核细胞占的PBMC的约6.2％，而由磁选分离的单核细胞占的PBMC的高达25％。统计分析表明，通过磁分离的单核细胞产生的比例分别显著升高（≥4倍）​​。数据表示为至少三个独立实验的平均值±标准差。使用学生氏t比较这两种方法（P≤0.0005）时-test显示单核细胞产率的一个显著差异的统计分析。 _content"FO:保together.within页=""&gt; 分离单核细胞贴壁和磁分离 1 不影响单核细胞活力 由上述的两种不同技术的单核细胞分离后，进行存活测定，以确保使用的方法并没有毒性和影响细胞的活力。 图2显示的数据提出了通过两种方法粘附或通过磁选分离出单核细胞存活的百分比的平均值。利用在隔离当日的台盼蓝排除法活细胞进行计数。两个隔离方法之间的单核细胞百分比比较生存能力透露，这两个群体之间的差异无统计学显著。此外，通过坚持孤立可行的单核细胞的百分比平均为91.75％±1.66和可行单核细胞百分比的平均值由MAGN隔离客位分离为87.4％±2.75。这些存活数据来自至少三个独立的实验进行评估。使用学生氏t检验测定统计学显着性。 从单核细胞细胞磁选分离显示细胞增殖相比增长从单核细胞通过细胞贴壁法收购 MTT被用作比色测定法来测量细胞增殖。在活细胞中，MTT法的黄色四唑颜色被减少到紫色的甲，这是很容易使用分光光度计进行测定。在图3中呈现的数据证明，如通过在第0天的吸光度增加（粘附方法测定的单核细胞分离的两个过程导致较高的细胞增殖一段7天:0.22±0.02 对磁法:0.38±00.02），3日（贴壁法:0.28±0.02，磁法:0.55±0.05）和7天（贴壁法:0.36±0.01，磁= 0.66±0.006）。然而，不同于XTT分析（数据未示出），MTT测定法揭示MDDCs的细胞增殖通过磁选分离的单核细胞相比，单核细胞在第0天（p值= 0.003），第3天通过粘附方法获得MDDCs一个显著增加（p值= 0.006），和第7天（p值= 0.002）。细胞的增殖也被发现是天0和3（p值= 0.003）之间显著不同，并通过磁分离纯化天0和7的组从单核细胞MDDCs的内（P &lt;0.001）之间。此外，观察到0天和在MDDCs7天之间细胞的增殖，从单核细胞显著差异通过粘接（p值= 0.008）纯化。 MTT测定，使用从三个不同的实验取得MDDCs进行。使用ANOVA和学生t检验，p≤0.05瓦特测定统计学显着性如考虑显著和p值在图上指出，除非无统计学意义被发现。 通过的CD11c和CD14细胞表面染色MDDCs表征 培养七天与IL-4和GM-CSF的单核细胞后，从由加入和磁分离方法分离的单核细胞获得的MDDCs表征于图4。表征揭示低单CD14 + / CD11c-表型或单纯单核细胞群体在两种方法中，粘附= 1％±1.0对比磁性= 1％±0.0和高的总的CD11c +表型，粘附= 72％±11相对于磁性= 79％±19。然而，当总的CD14 +群体进一步分析，有高产率细胞双阳性与磁选给予较多的CD11c和CD14双阳性的这两种方法的CD11c和CD14人口相比时的粘附方法，粘附= 49％±7与磁性= 73％±15。虽然，有通过磁分离，在CD11c阳性和CD14阴性人口MDDCs的双阳性表型MDDCs的较高产率更高在坚持的方法;粘附= 23％±7与磁性= 6％±7。在图4B中 ，每个单独的标记物的平均荧光强度（MFI）在门控人口搜索并显示CD14的高强度在CD14 +群体，两者的等效强度的CD11c和CD14双阳性人群和CD11c的+和CD14-人口高强度的CD11c。总之，即使磁隔离给出的CD11c + / CD14 +细胞的比例更高，这可能是早期分化MDDCs尚未下降的单核细胞标记（CD14），粘附方法给出的CD11c更高百分比+ / CD14-，这可以是晚期分化MDDC人口。在ADDIT离子，进行DAPI染色以确认存活力的结果，并确保在带电MDDCs进行表征。 DAPI阴性/活细胞的百分比（粘附= 76±7与磁性= 67±1）不是两种方法确认这两种方法在培养7天不影响MDDCs的存活率之间显著不同。 <img alt="图1" src="/files/ftp_upload/54296/54296fig1.jpg" /> 图1. 单核细胞通过产量磁分离较高相比于单核细胞通过产量贴壁法。约6.2％的单核细胞被利用贴壁法，而〜用磁选法获得的单核细胞25％的外周血单个核细胞分离出来。数据表示为至少三个独立实验的平均值±标准差。比较这两种方法时的统计分析使用学生t检验表明单核细胞产率的一个显著差异（P = 00005）。 <a href="https://www.jove.com/files/ftp_upload/54296/54296fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54296/54296fig2.jpg" /> 图2. 单核细胞被分离，并坚持通过磁分离，不影响单核细胞的可行性。通过坚持孤立可行的单核细胞百分比的平均值为91.75％±1.66和通过磁选分离出单核细胞存活的百分比平均为87.4％± 2.75。数据表示为平均值±标准差的至少三个独立实验的存活细胞的百分比。比较两个纯化方法时，细胞存活率没有观察到显著差异。 <a href="https://www.jove.com/files/ftp_upload/54296/54296fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html><img alt="图3" src="/files/ftp_upload/54296/54296fig3.jpg" /> 从单核细胞通过磁选分离 图3. 细胞显示在细胞增殖相比增长由贴壁法从单核获得的细胞。观察到这两种方法在两个天3和7天的吸光度A显著上升比较值时，针对其各自的第0天。另外控制，ANOVA和学生t检验比较这两种方法（p≤0.05）时也显示在MTT摄取吸光度显著差异。数据表示为三个独立实验的平均值±标准差。 <a href="https://www.jove.com/files/ftp_upload/54296/54296fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 96 / 54296fig4.jpg"/&gt; 图4. 表征的CD11c和CD14细胞表面染色。A）条形图MDDCs代表不同的群体（％选通的单细胞） 的 。首先，DAPI-（活着）细胞从总的单个细胞门控。然后，CD14 + / CD11c-，CD11c的+，CD11c的+ / CD14 +的CD11c和+ / CD14-从DAPI-（活的）人口门。的CD11c +群体是两个区域的CD11c + / CD14 +和CD11c的+ / CD14-。B）的代表性的曲线图表示; C）细胞的代表性散点图的细胞的每一个细胞亚群的平均荧光强度（MFI）值两者的CD11c和CD14标记的总和用的CD11c-APC和CD14-APCcy7门上DAPI阴性（活着）细胞群为磁性及坚持的方法。属于每个细胞亚群的单个细胞的代表性图像库（在散点图中蓝色选中）时，表面CD11c + / CD14-（门控橙色），的CD11c + / CD14 +（门控在红色），CD14 +（紫色门控）。在单细胞的图像画廊，红色代表的CD11c标记APC和黄色代表标记APCcy7 CD14。在散点图，选择门人口的颜色是随机的，不相关的实际细胞图像。 <a href="https://www.jove.com/files/ftp_upload/54296/54296fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

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

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

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

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

characterization-human-monocyte-derived-dendritic-cells-imaging-flow

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.4K 次观看.  Florida International University. This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

科研

免疫与感染

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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

从全血人单核细胞来源的树突状细胞的生成

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

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工程

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

原人类单核细胞的隔离、转染和文化

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

使用唾液酸酶处理从分离的 PBMC 中去除唾液酸化单核细胞衍生的 DC

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

作者聚焦:使用基于唾液酶的细胞表面酶处理树突状细胞成熟 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

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

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原人类单核细胞的隔离、转染和文化

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成