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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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD14++blood+monocytes+can+differentiate+into+functionally+mature+CD83++dendritic+cells.">CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.</a> Proc Natl Acad Sci. 93, 2588-2592 (1996).</li><li>Caux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B70/B7-2+is+identical+to+CD86+and+is+the+major+functional+ligand+for+CD28+expressed+on+human+dendritic+cells.">B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells.</a> J Exp Med. 180, 1841-1847 (1994).</li><li>Caux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+human+dendritic+cells+through+CD40+cross-linking.">Activation of human dendritic cells through CD40 cross-linking.</a> J Exp Med. 180, 1263-1272 (1994).</li><li>Fujii, S. i., Liu, K., Smith, C., Bonito, A. J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Linkage+of+Innate+to+Adaptive+Immunity+via+Maturing+Dendritic+Cells+In+Vivo+Requires+CD40+Ligation+in+Addition+to+Antigen+Presentation+and+CD80/86+Costimulation.">The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation.</a> J Exp Med. 199, 1607-1618 (2004).</li><li>Mohammadi, A., Mehrzad, J., Mahmoudi, M., Schneider, M., Haghparast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+culture+and+maturation+on+human+monocyte-derived+dendritic+cell+surface+markers,+necrosis+and+antigen+binding.">Effect of culture and maturation on human monocyte-derived dendritic cell surface markers, necrosis and antigen binding.</a> Biotech Histochem. 90, 445-452 (2015).</li></ol>

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?...

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。

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		<tr height="34">
			<td height="34" style="height:34px;width:535px;">Ficoll-Paque</td>
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			<td style="width:231px;">17-5442-03</td>
			<td style="width:443px;">Must be used at room temperature</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">ACK Lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Antibiotic-Antimycotic (100X)</td>
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			<td>15240-062</td>
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		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RoboSep buffer</td>
			<td>StemCell</td>
			<td>20104</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
			<td>StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FITC-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
			<td>Biotek</td>
			<td>7131000</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">XTT Sodium salt bioreagent (XTT)</td>
			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0302</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phenazine Methosulfate (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>P9626-1G</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Inactivated (HI) Human Serum</td>
			<td>Chemicon</td>
			<td>S1-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
			<td></td>
		</tr>
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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...

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 ...

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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

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

科研

免疫与感染

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 ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

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 ...

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 ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

siRNA 电穿孔以在单纯疱疹病毒类型 1-感染单细胞衍生的丹突细胞中调制自噬

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...