笔者想在过去的几年里，感谢每棵Schaffrinski太太对她始终以卓越的技术援助。 这项工作是通过德国研究会（DFG）的联合调研组942（FOR942）内及医学院，乔治 - 月 - 哥廷根大学的研究项目资助。

1，制备无菌人AB血清<ol><li>收集4-5袋新鲜冰冻血浆（FFP）。店内包包在-20°C，直到足够的包包已收集。 </li><li>解冻袋孵育30分钟，在56℃水浴灭活补体和纤维蛋白去除。 </li><li>彻底消毒的包装袋的外侧，并在等离子体转移到50ml试管。 </li><li>离心机在3000×g离心15分钟，在室温下，以除掉沉淀物和残余的血小板。 </li><li>汇集所有上清液并弃去小球。 </li><li>在15ml试管等分试样的血清样品并储存在-20℃。 注意：可替换地，可商购的热失活的正常的人AB血清可以使用。 </li></ol> 2，分离单核细胞为方便离心机的平衡，建议处理两个并联的血沉棕黄层。不过，以CA重新使用分开的材料对于每个供体，而不是混合的细胞。万一血沉棕黄层不能容易地获得，可用于400毫升肝素化外周血代替。 <ol><li>仔细消毒塑料袋含有棕黄色大衣，每个棕黄色大衣的内容传送到两个50 ml离心管。 </li><li>对于每一个棕黄色的外套填写3 50ml试管用15毫升聚蔗糖溶液（1.077克/毫升）。聚蔗糖应在室温下制备。 </li><li>层30-35毫升的血沉棕黄层血液上的第一密度梯度的聚蔗糖溶液的顶部。要小心，以防止混合这两层缓慢且小心地做到这一点。 </li><li>离心400 XG不带刹车30分钟，在室温下。 </li><li>为每个梯度收集的外周血单核细胞（PBMC），其位于两相，用塑料巴斯德吸管，并转移到50毫升管之间的白圈。 </li><li>填写各管，用PBS-EDTA（1毫米）到40毫升总。 </li><li>离心300 XG在室温下10分钟不带刹车。 </li><li>吸出上清液，用40毫升的PBS-EDTA（1毫米）洗一遍沉淀。 </li><li>对于每个供体池中的粒料在20毫升的RPMI-1640无酚红+10％FCS中。 </li><li>准备第二密度梯度的等渗珀的解决方案：对于两个供体混合23.13毫升珀溶液（密度：1.131克/ ml）的50毫升管1.87毫升10倍的PBS。然后转移23毫升此溶液到新的50ml管中，并添加27毫升的RPMI-1640与酚红+10％FCS中，得到46％的等渗的Percoll溶液。上述珀应在室温下制备。 </li><li>对于每个供体转移将25ml制备的Percoll溶液至50ml管中并层上的Percoll溶液的顶部在2.9中制备的PBMC中的溶液）。要小心，这样做很慢的ð仔细，两层往往容易混淆。如果做得正确的两个阶段可能会因自己的色差来区分。 </li><li>离心550 XG不带刹车30分钟，在室温下。 </li><li>为每个梯度收集单核细胞，位于该两相，用塑料巴斯德吸管，并转移到50毫升管之间的白圈。 </li><li>填充每个管子用PBS-EDTA（1毫米）至总体积为50ml。 </li><li>离心400 XG在室温下10分钟不带刹车。 </li><li>吸出上清液，并用酚红+10％FCS中悬浮的颗粒在20毫升的RPMI-1640。 </li></ol> 3，单核细胞分化的巨噬细胞<ol><li>确定在1:10稀释在台盼蓝分离的单核细胞的数量。只算大，常有不规则形状的细胞，这是单核细胞。不要指望较小，圆形细胞是淋巴细胞。 ÑOTE：单核细胞数没有确定太精确，因为它们只给出一个关于多少和哪些铁氟龙被覆袋（小的或大的）必须被用于细胞的分化提示。 </li><li>从一个供体1.0〜1.5×10 8单核细胞，种子细胞在一个大的铁氟龙涂层的细胞培养袋。对于每个袋准备由174毫升的RPMI-1640，2％人AB血清（如制备中第1项），1％青霉素/链霉素和2.5毫微克/毫升M-CSF（总体积：180毫升）的培养基中。 <ol><li>从一个供体3.0〜5.0×10 7的单核细胞，种在小特氟龙涂层的细胞培养袋细胞。对于每个袋子制备的培养基组成的28.5毫升的RPMI-1640，2％人AB血清（如制备中第1项），1％青霉素/链霉素和2.5毫微克/毫升M-CSF（总体积：30毫升）中。 注：如果如 8.0×10 7单核细胞获得，我们建议这些种子在台湾ö较小体积袋与每个4.0×10 7个细胞。代替M-CSF的，单核细胞也可以被区分具有相同浓度（2.5纳克/毫升）粒细胞巨噬细胞集落刺激因子（GM-CSF）。 </li></ol></li><li>加入细胞悬浮液（20毫升）中所制备的培养基中，并仔细混匀。如果两包从一个供体制备，加20毫升的RPMI-1640的细胞悬浮液，然后加入一半的悬浮液的每一个介质制备。 </li><li>可选：如果要判断单核细胞制剂一直保持无菌，撕裂细胞悬浮液一滴在血琼脂平板上，孵育过夜，在37℃。 </li><li>拧紧的50毫升注射器perfusor鞘上铁氟龙涂层的细胞培养袋的插头，并与制备的细胞悬液填充它。 </li><li>拔掉注射器推剩余空气从袋子中。关闭包里带一节闭幕锥。 </li><li>在37孵育袋6-7天76;℃，5％CO 2中。 </li></ol> 4，巨噬细胞收获<ol><li>将FEP特氟龙涂层的细胞培养袋与所述分化的巨噬细胞在冰上放置至少1小时，以促进细胞的分离（达3小时是可能的孵育）。确保该袋的整个表面覆盖有冰。 </li><li>拉包以最小的压力10倍以上书桌/板的边缘。 </li><li>小心消毒袋的外面，并删除在截止锥体。 </li><li>拧紧的50ml注射器上的袋的插头，取出细胞悬浮液，并将其转移至50ml管中。 </li><li>离心机在400×g离心10分钟，在室温下，以降速的细胞。 </li><li>吸出上清液，并从一个袋子凝聚颗粒在10ml的RPMI-1640 +10共％FCS中。 </li><li>测定细胞数用血细胞计数器（1：4-1：10稀释在锥虫蓝）。只算大的圆形细胞。 注：微分的铁氟龙被覆袋巨噬细胞后，可以存在残留的细胞，例如淋巴细胞，这取决于供体和在单核细胞分离的质量。 </li><li>重用铁氟龙被覆袋，用70％乙醇洗两次他们以去除残余的细胞。它们用50毫升70％乙醇填充孵育过夜。洗袋3倍用无菌PBS，将它们包装在消毒纸，并用标准程序在高压釜中灭菌。 注意：所述的细胞培养袋可以在不中的巨噬细胞产率的损失可以重复使用多次。然而，我们决定放弃袋10使用后，他们开始泄漏之前。 </li><li>在RPMI-1640 +10％FCS的后续实验培养的巨噬细胞。如果降低血清浓度是必要的，培养在RPMI-1640，用1％FCS的也是可能的。种子和细胞孵育3-4小时。在此期间后，请注意，巨噬细胞附着到细胞培养皿而表面任何污染的细胞（ 即红细胞，淋巴细胞和树突状细胞）留在悬浮液中。用PBS至少洗涤细胞两次以除去非粘附细胞。 </li><li>分离从细胞培养皿中用于进一步实验培养的巨噬细胞，小心刮掉它们关闭的表面上。备选地，孵育菜20分钟在冰上，吸出培养基，并加入1 ml无酶细胞解离缓冲液。 5分钟后，在37℃下，加入4毫升的PBS，并分离细胞通过上下吹打至少3分钟。 </li></ol>

<ol>
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J., Bachli, E. B., Kurrer, M. O., Schoedon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5A/CaMKII+signaling+contributes+to+the+inflammatory+response+of+macrophages+and+is+a+target+for+the+antiinflammatory+action+of+activated+protein+C+and+interleukin-10.">Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10.</a> Arterioscl Throm Vas. 28, 504-510 (2008).</li></ol>

作者什么都没有透露是没有利益冲突存在。

巨噬细胞是先天免疫系统的重要效应细胞，并显示在免疫调节，抗原呈递和组织稳态的重要功能。由于其显着的可塑性，所以能与他们的表型的变化作出反应，以不同的刺激。然而，迄今为止，很多关于巨噬细胞的极化数据在鼠系统获得的，虽然有报道显示，只有约50％的巨噬细胞极化的标记可以直接翻译从小鼠到人类16。因此，我们在这里提出的方法，以获得足够数量和纯度原代人类巨噬细胞中，而不需要昂贵的材料， 例如 ，MACS磁珠或逆流离心淘析设备。 我们的方法是基于单核细胞从外周血单个核细胞的分离和其随后的分化的巨噬细胞中的FEP在低conce的存在特氟龙涂层的细胞培养袋M-CSF 11-13 ntrations。而单核细胞弥补不足5％至10％的外周血白细胞在人类中，在刺激时它们被招募到外周部位在那里分化到驻地组织巨噬细胞或树突状细胞17。的细胞因子M-CSF是单核细胞生存的重要，并驱动其分化为巨噬细胞18,19。到目前为止，它被选择用于单核细胞分化的M-CSF浓度范围高达100纳克/毫升，然而，在我们的协议中，我们能够获得足够数量的成熟的巨噬细胞与仅2.5纳克/毫升12 M-CSF浓度20。此外，细胞是在FEP特氟龙涂层的细胞培养袋其中促进巨噬细胞的分离和随后播种在限定的细胞数进行培养。由于袋可以重复使用多次，这进一步减小了隔离处理的费用。 通过这个过程得到的巨噬细胞对于CD45，CD14，CD11b的，CD11c的高度肯定，并显示甘露糖受体CD206的主张纯粹的，成熟的巨噬细胞21,22人群的表达。特别高的CD14表达是典型的巨噬细胞分化中M-CSF 23的存在。细胞接种后，他们显示了快速遵守塑料表面的一些细胞呈现出典型的纺锤状形态，而其他表现一个煎鸡蛋的表型。这是根据观测来自其他作者的18,22,24。 据报道，在M-CSF存在下的单核细胞分化导致M2极化巨噬细胞16,25。然而，分离由我们的协议的巨噬细胞仍然能够以大范围的刺激包括暴露于肿瘤细胞来源的微泡和共培养的肿瘤细胞26,27或暴露于LPS的响应它们与感应亲 ​​的反应炎症基因，如IL-1和＃946;，TNFα，Wnt5a的，或者被认为是典型的货币供应量M1极化巨噬细胞3,5,28不同的基质金属蛋白酶。 总之，单核细胞由双密度梯度离心和向在FEP特氟龙涂层的细胞培养袋结果在高数量的巨噬细胞巨噬细胞随后的分化，而不需要技术上的困难或昂贵的程序隔离。所获得的巨噬细胞可用于随后的分析通过LPS对共培养的肿瘤细胞，从经典的活化。

巨噬细胞- -从单核细胞谱系和他们的终末分化衍生的细胞表现出显着的可塑性在考虑到它们的生物学功能，从而导致其在如发育，组织修复和免疫1等不同的过程的参与。后者是由于它们的吞噬细胞和其在先天免疫和适应性免疫应答2之间的十字路口放置的巨噬细胞的抗原呈递能力。然而，它们的能力，以分泌细胞因子，趋化因子，生长因子，以及其他信号分子1不仅增强了其免疫调节功能，而且还可以作为其附加功能的基础。试图镜中的非微生物介导的病症的上下文中这些不同的活化步骤，导致了M1和M2的类别3。虽然这种分类是不完整的，它允许巨噬细胞生物学的一个基本的了解。 由于这些多方面的能力一点也不奇怪，巨噬细胞有很多条件，在某种程度上涉及组织重建或炎症有关。下一步，在入侵的病原体4-6识别和清除它们的基础性作用，巨噬细胞也日益成为关注的焦点在动脉粥样硬化，纤维化，肥胖和癌症7-10。对人类巨噬细胞的产生可复制的方法，因此，对这些疾病的认识至关重要。这里我们提出了如先前11描述的基础上，从健康供体的外周血人单核细胞中由双密度梯度离心法分离的方法。为了便于向巨噬细胞分化，分 ​​离的单核细胞在低浓度的M-CSF和正常人血清12的存在。以减轻进一步的处理和收获细胞，分化是在气体可渗透的FEP聚四氟乙烯涂层的细胞培养袋与疏水表面12-15。所得到的静止巨噬细胞可经受广泛的测定中，因为它们仍然能够在任一M1或M2状的方式响应的​​。的单核细胞的分离和随后的分化的替代方法，例如磁激活细胞分选（MACS）或逆流离心淘洗（CCE）具有关于收率，成本和所需时间一定的局限性。本文描述的协议提供，它可以进行与标准实验室设备，而不需要特殊的试剂（ 例如 ，MACS磁珠）或设备（ 例如 ，CCE装置），并允许对大量的细胞进行处理的优点。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>antibodies for immunophenotyping</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>for example: CD11c-PE (IM1760), CD45-FITC (7782), IgG1-PE isotype control (A07796), IgG1-FITC isotype control (A07795)</td>
		</tr>
		<tr>
			<td></td>
			<td>BioLegends</td>
			<td></td>
			<td>for example: CD14-FITC (325603), CD206-PE (321105)</td>
		</tr>
		<tr>
			<td>Axiovert 200M microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>calcein-AM</td>
			<td>AnaSpec</td>
			<td>89201</td>
			<td></td>
		</tr>
		<tr>
			<td>combi-stopper closing cones</td>
			<td>Braun</td>
			<td>4495101</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS, w/o Ca and Mg</td>
			<td>Pan biotech</td>
			<td>P04-36500</td>
			<td>for PBS-EDTA (1 mM) add 1 ml 0,5 M EDTA per 500ml PBS</td>
		</tr>
		<tr>
			<td>10x PBS, w/o Ca and Mg</td>
			<td>Invitrogen</td>
			<td>14200-067</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Titriplex III)</td>
			<td>Merck</td>
			<td>1084211000</td>
			<td>prepare a 0,5 M solution in H2O, use a sterile filter</td>
		</tr>
		<tr>
			<td>cell dissociation buffer (enzyme-free, PBS-based)</td>
			<td>Gibco</td>
			<td>13151-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen</td>
			<td>10091148</td>
			<td>heat-inactivated</td>
		</tr>
		<tr>
			<td>Ficoll (density 1.077g/ml)</td>
			<td>Biochrom AG</td>
			<td>L6115</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCanto II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse FITC</td>
			<td>santa cruz</td>
			<td>sc-2010</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E.coli</td>
			<td>Sigma</td>
			<td>L8274</td>
			<td>final conc: 100 ng/ml</td>
		</tr>
		<tr>
			<td>Multifuge 3 L-R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Biochrom AG</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (density 1,131g/ml)</td>
			<td>GE Healthcare</td>
			<td>17-0891-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusor syringe 50ml</td>
			<td>Braun</td>
			<td>8728844F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-TRITC</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>resuspend in methanol (c = 0,1 mg/ml)</td>
		</tr>
		<tr>
			<td>Plastic Disposable Pasteur Pipettes</td>
			<td>LVL technologies</td>
			<td>2655181</td>
			<td></td>
		</tr>
		<tr>
			<td>rh M-CSF</td>
			<td>ImmunoTools</td>
			<td>11343117</td>
			<td>Resuspend in 500&#181;l sterile H2O (c = 100 ng/&#181;l), aliquot</td>
		</tr>
		<tr>
			<td>RPMI-1640 with Phenol Red</td>
			<td>Gibco</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 without Phenol Red</td>
			<td>Gibco</td>
			<td>11835-063</td>
			<td></td>
		</tr>
		<tr>
			<td>sterilization paper</td>
			<td>VP group</td>
			<td>3KFKFS230116</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue stain (0,4% w/v)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>FEP Teflon-coated cell culture bag, small</td>
			<td>CellGenix</td>
			<td>72-C</td>
			<td></td>
		</tr>
		<tr>
			<td>FEP Teflon-coated cell culture bag, large</td>
			<td>CellGenix</td>
			<td>197-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags

人巨噬细胞参与了众多的病理过程，从传染病到癌症。因此，他们构成一个有价值的工具来了解这些疾病的基本机制。因此，我们提出了一个简单的协议，用于从血沉棕黄层人体单核细胞，随后分化过程导致高的巨噬细胞产率的分离。该技术主要依赖于常用的实验室设备，从而提供了成本和时间有效的方式来获得大量人类巨噬细胞中。简言之，从健康献血员的血沉棕黄层经受双密度梯度离心从外周血收获的单核细胞。这些单核细胞，然后培养中的巨噬细胞集落刺激因子（M-CSF）存在下氟化乙烯丙烯（FEP），聚四氟乙烯涂覆的细胞培养袋。分化的巨噬细胞可以容易地收获并用于后续的研究和功能如说。质量控制和隔离和分化步骤验证的重要方法会在协议中加以强调。总之，这里所描述的方案使科学家能够定期地和可重复性地分离人的巨噬细胞，而无需成本密集型的​​工具。此外，疾病模型可以研究在人类的同源规避系统利用小鼠巨噬细胞。

使用聚蔗糖第一密度梯度离心得到含有的PBMC（ 图1A）， 即淋巴细胞和单核细胞白相间。这可以通过一个五月Gruenwald染色所收集的细胞，同时显示了高的核/质比（典型的淋巴细胞）的（ 图1B和C）进行确认和bean-或环状的细胞核（典型的单核细胞）。当这些细胞，然后加载到用Percoll密度的第二密度梯度，将单核细胞可以从淋巴细胞进一步分离和再次出现，为白色相间（ 图1D-F）。对于每个棕黄层所描述的双密度梯度离心例行得到150±40×10 6单核细胞可以分化为每棕黄层70±30×10 6个巨噬细胞（ 图2）。平均收益率巨噬细胞从20个独立的准备占总分离的单核细胞47±14％。 Percoll梯度离心后仍然有可能存在于它是依赖于供血者，以及在隔离过程的准确性的制备一些残留的非单核细胞。然而，在6-7天的分化阶段后，该制剂主要由成熟的巨噬细胞（ 图3），其可由于它们粘附到塑料表面上，即不是由无规污染细胞共有的特征（ 图4A进一步富集的和B）。镀后，大多数巨噬细胞表现出典型的“荷包蛋”的形态，同时也存在着细胞伸出主轴样表型（ 图4C和D）。这是由它们的细胞质和粘附群内的F-肌动蛋白分布的镜像。分化细胞的特征在于CD45，CD14，CD16，CD206（甘露糖受体），细胞CD11b的，哪些是成熟的巨噬细胞（ 图5）的典型标志物的表达CD11c的。 CD11b的存在指出针对所支持的事实，即所述细胞是阴性的树突状细胞的标记CD209（DC-SIGN），主要为树突分化。 分化过程后，将细胞保持在功能和代谢活性为约5-7天（ 图6），因为它可以通过钙黄绿素AM染色和其占用从肿瘤细胞脱落的细胞外囊泡的能力来显示。此外，细胞仍然可以激活如图所示，例如 ，用于与脂多糖（LPS）的刺激作用导致的几种促炎基因（ 图7）中的表达。 <img alt="图1" fo:content-width="5in"SRC =“/文件/ ftp_upload / 51554 / 51554fig1highres.jpg”WIDTH =“500”/&gt; 后双密度梯度离心图1，外观和PBMC-的组合物和单核细胞层。照片表示的Ficoll梯度和等渗Percoll密度离心后（D）的单核细胞相后（A）的PBMC的波段。五月，Gruenwald染色的（B，C）PBMC部分的细胞离心涂片准备，剩下的（E，F）的单核细胞。比例尺= 200 B和E微米= 50微米，C和F。 <img alt="图2" fo:content-width="3in" src="/files/ftp_upload/51554/51554fig2highres.jpg" width="300" /> 图2。产量单核细胞和巨噬细胞的，孤立的单核细胞代表的计数和20白膜层PR巨噬细胞eparations。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51554/51554fig3highres.jpg" width="600" /> 图3显微照片和单核细胞和巨噬细胞。的单核细胞悬浮液相衬显微镜的前（A）和之后（B）中的巨噬细胞分化的细胞大小的测量 。相应的单核细胞（C）和巨噬细胞（D）的单元尺寸直方图。比例尺=100μm左右。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51554/51554fig4highres.jpg" width="600" /> 图4形态和附着的巨噬细胞的细胞骨架组织。贴壁的巨噬细胞的前（A）相衬显微镜和（b）后除去未adheren的T细胞。在粘附的，非刺激的巨噬细胞（C，D）的鬼笔环肽-TRITC丝状肌动蛋白的染色。比例尺为100μm的交流，在D 20微米<a href="https://www.jove.com/files/ftp_upload/51554/51554fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51554/51554fig5highres.jpg" width="500" /> 图5。免疫表型分化的巨噬细胞。6天后分化的铁氟龙涂层的细胞培养袋流式细胞仪的巨噬细胞分析（以红色表示）。相应的同型对照显示为灰色。 <a href="https://www.jove.com/files/ftp_upload/51554/51554fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 图6：由巨噬细胞摄取肿瘤细胞的微泡。博览会PKH26标记（红色荧光）的肿瘤细胞来源的微泡后贴壁的巨噬细胞的显微照片。图像被叠加到相应的（A）明场或（B）的细胞内染色的存活率染料钙黄绿素AM。比例尺= 100微米。 <a href="https://www.jove.com/files/ftp_upload/51554/51554fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51554/51554fig7highres.jpg" width="500" /> stimu图7后上调的IL-1β，Wnt5a的，TNFα，IL-6，MMP-2，MMP-7，和MT1-MMP巨噬细胞用LPS（100毫微克/毫升）24小时。基因表达的特征研通过定量RT-PCR从总RNA样品（A）和归一化上HPRT1和GNB2L1表达测定。所示的值是相比于未处理的对照倍数变化（平均值±SD，N ​​= 5，* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001）。下LPS刺激的TNFα和IL-6的诱导通过ELISA（B），进一步证实了（平均值±SD，* P &lt;0.05）。

Watch this Scientific Journal Video about Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags at JoVE.com

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

我们提出了一个简单而有效的协议对人类巨噬细胞的产生。血沉棕黄层用双密度梯度离心法处理，并分离单核细胞，然后分化为巨噬细胞中的特氟龙涂层的细胞培养袋。这最大限度地提高巨噬细胞的产量，并促进细胞收集在后续实验。

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 ...

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69.3K 次观看.  University Medical Center Göttingen. 我们提出了一个简单而有效的协议对人类巨噬细胞的产生。血沉棕黄层用双密度梯度离心法处理，并分离单核细胞，然后分化为巨噬细胞中的特氟龙涂层的细胞培养袋。这最大限度地提高巨噬细胞的产量，并促进细胞收集在后续实验。 

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

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

科研

免疫与感染

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

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. ...