Name: in vitro stimulation and visualization of extracellular trap release in differentiated human monocyte-derived macrophages
Uploaded: 2019-11-01T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages at JoVE.com

这项工作得到了永久IMPACT赠款（IPAP201601422）和诺和诺德基金会生物医学项目赠款（NNF17OC0028990）的支持。YZ 还感谢悉尼大学获得澳大利亚研究生奖。我们要感谢帕特·皮桑萨拉基特先生和摩根·琼斯女士在单核细胞隔离和组织培养方面给予的帮助。

HMDM与血库提供的人体布漆制剂分离，并经悉尼当地卫生区道德认证。
1. HMDM文化
<ol><li>利用市售制剂分离淋巴细胞，从健康人类捐献者外周血中制备的单核细胞，然后逆流离心性电镀19， 20.</li>
	<li>确认单核细胞的存在，通过细胞旋转和染色与修饰的吉姆萨染色单细胞表征19。</li>
	<li>在无菌条件下，使用不含血?...

<ol>
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F., 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=Neutrophil+extracellular+traps+contain+calprotectin,+a+cytosolic+protein+complex+involved+in+host+defense+against+Candida+albicans.">Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.</a> PLoS Pathogens. 5 (10), 1000639 (2009).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+suicide:+why+neutrophils+die+to+make+NETs.">Beneficial suicide: why neutrophils die to make NETs.</a> Nature Reviews in Microbiology. 5 (8), 577-582 (2007).</li><li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews in Immunology. 18 (2), 134-147 (2018).</li><li>Keshari, R. S., 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=Cytokines+induced+neutrophil+extracellular+traps+formation:+implication+for+the+inflammatory+disease+condition.">Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition.</a> PLoS One. 7 (10), 48111 (2012).</li><li>Rayner, B. S., 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=Role+of+hypochlorous+acid+(HOCl)+and+other+inflammatory+mediators+in+the+induction+of+macrophage+extracellular+trap+formation.">Role of hypochlorous acid (HOCl) and other inflammatory mediators in the induction of macrophage extracellular trap formation.</a> Free Radical Biology and Medicine. 129, 25-34 (2018).</li><li>Gunzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traps+and+hyperinflammation+-+new+ways+that+neutrophils+promote+or+hinder+survival.">Traps and hyperinflammation - new ways that neutrophils promote or hinder survival.</a> British Journal of Haematology. 164 (2), 189-199 (2014).</li><li>Knight, J. S., 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=Peptidylarginine+deiminase+inhibition+reduces+vascular+damage+and+modulates+innate+immune+responses+in+murine+models+of+atherosclerosis.">Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.</a> Circulation Research. 114 (6), 947-956 (2014).</li><li>Megens, R. T., 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=Presence+of+luminal+neutrophil+extracellular+traps+in+atherosclerosis.">Presence of luminal neutrophil extracellular traps in atherosclerosis.</a> Thrombosis and Haemostasis. 107 (3), 597-598 (2012).</li><li>Doring, Y., Weber, C., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Footprints+of+neutrophil+extracellular+traps+as+predictors+of+cardiovascular+risk.">Footprints of neutrophil extracellular traps as predictors of cardiovascular risk.</a> Arteriosclerosis Thrombosis and Vascular Biology. 33 (8), 1735-1736 (2013).</li><li>Goldmann, O., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+world+of+extracellular+traps:+not+only+neutrophils+but+much+more.">The expanding world of extracellular traps: not only neutrophils but much more.</a> Frontiers in Immunology. 3 (420), 1-10 (2012).</li><li>Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+traps+and+macrophages:+new+roles+for+the+versatile+phagocyte.">Extracellular traps and macrophages: new roles for the versatile phagocyte.</a> Journal of Leukocyte Biology. 97 (6), 1023-1035 (2015).</li><li>Pertiwi, K. R., 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=Extracellular+traps+derived+from+macrophages,+mast+cells,+eosinophils+and+neutrophils+are+generated+in+a+time-dependent+manner+during+atherothrombosis.">Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis.</a> Journal of Pathology. 247 (4), 505-512 (2019).</li><li>Okubo, K., 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=Macrophage+extracellular+trap+formation+promoted+by+platelet+activation+is+a+key+mediator+of+rhabdomyolysis-induced+acute+kidney+injury.">Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury.</a> Nature Medicine. 24 (2), 232-238 (2018).</li><li>Boeltz, S., 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=To+NET+or+not+to+NET:current+opinions+and+state+of+the+science+regarding+the+formation+of+neutrophil+extracellular+traps.">To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.</a> Cell Death and Differentiation. 26 (3), 395-408 (2019).</li><li>Doster, R. S., Rogers, L. M., Gaddy, J. A., Aronoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Extracellular+Traps:+A+Scoping+Review.">Macrophage Extracellular Traps: A Scoping Review.</a> Journal of Innate Immunology. 10 (1), 3-13 (2018).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+PMA-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS One. 5 (1), 8668 (2010).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> Journal of Immunology. 172 (5), 2731-2738 (2004).</li><li>Brown, B. E., Rashid, I., van Reyk, D. M., Davies, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+of+low-density+lipoprotein+results+in+the+time-dependent+accumulation+of+cholesteryl+esters+and+apolipoprotein+B-100+protein+in+primary+human+monocyte-derived+macrophages.">Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages.</a> FEBS Journal. 274, 1530-1541 (2007).</li><li>Garner, B., Dean, R. T., Jessup, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+macrophage-mediated+oxidation+of+low-density+lipoprotein+is+delayed+and+independant+of+superoxide+production.">Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independant of superoxide production.</a> Biochemical Journal. 301, 421-428 (1994).</li><li>Morris, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acid+ionization+constant+of+HOCl+from+5+&#176;C+to+35+&#176;C.">The acid ionization constant of HOCl from 5 &#176;C to 35 &#176;C.</a> Journal of Phyical Chemistry. 70, 3798-3805 (1966).</li><li>Pan, G. J., Rayner, B. S., Zhang, Y., van Reyk, D. M., Hawkins, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pivotal+role+for+NF-kappaB+in+the+macrophage+inflammatory+response+to+the+myeloperoxidase+oxidant+hypothiocyanous+acid.">A pivotal role for NF-kappaB in the macrophage inflammatory response to the myeloperoxidase oxidant hypothiocyanous acid.</a> Archives of Biochemistry and Biophysics. 642, 23-30 (2018).</li><li>Parker, H., Albrett, A. M., Kettle, A. J., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+associated+with+neutrophil+extracellular+traps+is+active+and+mediates+bacterial+killing+in+the+presence+of+hydrogen+peroxide.">Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide.</a> Journal of Leukocyte Biology. 91 (3), 369-376 (2012).</li></ol>

使用M1分化HMDM生成和可视化MET形成是一种新的体外模型，可能有助于研究这些巨噬菌体结构的潜在病理作用，特别是在慢性炎症下条件。它为刺激原发性人类巨噬细胞释放MET提供了强有力的方案，也可用于与人类单核细胞或鼠群细胞系相关的研究。HMDM刺激MET形成的该协议的成功实施取决于仔细的细胞培养和处理，以保持良好的细胞活力。这将提高所观察到的 MET 的范围和质量。为了提供更好的细?...

从嗜中性粒细胞释放的ET首先被确定为由细菌感染1触发的先天免疫反应。它们由DNA骨干组成，各种具有抗菌特性的颗粒蛋白被结合，包括嗜中性乳酸酶和骨髓过氧化物酶2。中性粒细胞（NETs）的主要作用是捕获病原体并促进其消除3。然而，除了ETs在免疫防御中的保护作用外，越来越多的研究也发现了疾病发病机制的作用，特别是在炎症驱动型疾病（即风湿性关节炎和动脉粥样硬化4。ET的释放可以由各种亲炎细胞因子触发，包括白细胞介素8（IL-8）和肿瘤坏死因子α（TNF+）5，6，并且ET的局部积累可以增加组织损伤，并引起亲炎反应7。例如，ET被牵连为在动脉粥样硬化8的发展，促进血栓形成9，和预测心血管风险10的因果作用。
现在人们认识到，除了嗜中性粒细胞外，其他免疫细胞（即乳腺细胞、嗜酸性粒细胞和巨噬细胞）也可以在暴露于微生物或亲炎刺激11、12时释放ETs。考虑到巨噬细胞在慢性炎症疾病的发展、调节和解决中的重要作用，这可能在巨噬细胞方面特别重要。因此，有必要更好地了解从巨噬细胞中释放ET与炎症相关疾病发展之间的潜在关系。最近的研究表明，在完整的人类动脉粥样硬化斑块和有组织的血栓13中存在MET和NET。类似地，METS也涉嫌通过调节炎症反应14来驱动肾损伤。然而，与嗜中性粒细胞相比，关于巨噬细胞的MET形成机制的数据有限。最近使用MET形成人类体外模型的研究表明，每种细胞类型（即缺乏巨噬细胞组蛋白的细胞）所涉及的途径存在一些差异。然而，一些已经表明，NET释放也可以发生在没有组蛋白的切口15。
该协议的总体目标是提供一种简单而直接的方法，在临床相关的巨噬菌体模型中评估MET释放。有许多不同的体外巨噬细胞模型，已用于研究METs（即，THP-1人类单细胞细胞系和各种鼠巨噬细胞系）16。这些模型存在一些限制。例如，THP-1单核细胞与巨噬细胞的分化通常需要一个启动步骤，例如添加醋酸磷酸酯（PMA），它本身激活蛋白质激酶C（PKC）依赖通路。此过程已知可触发 ET 释放4，并产生 THP-1 细胞的低基底 MET 释放。其他研究也突显了与PMA处理的THP-1细胞17相比，体内巨噬细胞在生物活性和炎症反应方面的差异。
同样，不同鼠群状细胞系的行为和炎症反应并不完全代表人类原发性巨噬细胞18的反应谱。因此，为了研究在临床环境中的巨噬细胞ET形成，主要人类单细胞衍生巨噬细胞（HMDMs）被认为是一个更相关的模型，而不是单细胞或鼠狼类似细胞的细胞系。
M1极化HMDM的ET释放已经证明，这些细胞暴露于许多不同的炎症刺激，包括骨髓过氧化物酶衍生的氧化氯酸（HOCl），PMA，TNF®和IL-86。此处描述的是一种将 HmDM 与 M1 表型极化的协议，并在暴露于这些炎症刺激时可视化后续 MET 释放。PMA被用作MET释放的刺激，以方便与以前使用中性粒细胞的研究进行比较。重要的是，HOCl、IL-8和TNF®也用于刺激MET释放，这被认为是体内炎症环境的更好模型。ET释放可视化的微观方法涉及使用SYTOX绿色染色活细胞培养物中的细胞外DNA，这是一种不透水的荧光绿色核酸染色，已成功应用于以前的中性粒细胞研究。此方法允许对 ET 释放进行快速和定性的评估，但它不适合作为 ET 释放范围的量化的独立方法。如果需要量化以比较不同治疗条件或干预措施产生的 ET 释放程度，则应使用替代方法。

<table>
	<tbody>
		<tr>
			<td>120Q broad spectrum fluorescent light source</td>
			<td>EXFO Photonic Solutions, Toronto, Canada</td>
			<td></td>
			<td>x-cite series</td>
		</tr>
		<tr>
			<td>Corning CellBIND Multiple Well Plate (12 wells)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3336</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Differential Quik Stain Kit (Modified Giemsa)</td>
			<td>Polysciences Inc.</td>
			<td>24606</td>
			<td>Characterisation of monocytes</td>
		</tr>
		<tr>
			<td>Hanks balanced salt solution (HBSS)</td>
			<td>Thermo-Fisher</td>
			<td>14025050</td>
			<td>For washing steps and HOCl treatment</td>
		</tr>
		<tr>
			<td>Hypochlorous acid (HOCl)</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Interferon gamma</td>
			<td>Thermo-Fisher</td>
			<td>PMC4031</td>
			<td>For M1 priming</td>
		</tr>
		<tr>
			<td>Interleukin 4</td>
			<td>Integrated Sciences</td>
			<td>rhil-4</td>
			<td>For M2 priming</td>
		</tr>
		<tr>
			<td>Interleukin 8</td>
			<td>Miltenyl Biotec</td>
			<td>130-093-943</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>59202C</td>
			<td>Added to culture media</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>Integrated Sciences</td>
			<td>tlrl-eblps</td>
			<td>For M1 priming</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield PoC AS</td>
			<td>1114544</td>
			<td>For isolation of monocytes</td>
		</tr>
		<tr>
			<td>Olympus IX71 inverted microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12- myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>For washing steps</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>SYTOX green</td>
			<td>Life Technologies</td>
			<td>S7020</td>
			<td>For MET visulaization</td>
		</tr>
		<tr>
			<td>TH4-200 brightfield light source</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td>x-cite series</td>
		</tr>
		<tr>
			<td>Tumor necrosis factor alpha</td>
			<td>Lonza</td>
			<td>300-01A-50</td>
			<td>For MET stimulation</td>
		</tr>
	</tbody>
</table>

in vitro stimulation and visualization of extracellular trap release in differentiated human monocyte-derived macrophages

嗜中性粒细胞释放细胞外陷阱（ETs）已被确定为导致慢性炎症相关疾病发展的一个因素。中性粒细胞（NETs）由DNA、组蛋白和各种颗粒蛋白（即骨髓过氧化物酶、电感酶和卡西普辛G）组成的网状物组成。其他免疫细胞，包括巨噬细胞，也可以产生ET;然而，这在体内发生的程度，以及巨噬细胞外陷阱（METs）是否在病理机制中发挥作用，尚未得到详细研究。为了更好地了解MET在炎症性病理中的作用，开发了一种方案，用于在体外可视化来自原发性人类巨噬细胞的MET释放，在免疫荧光实验中也可以加以利用。这允许进一步描述这些结构，并将其与从嗜中性粒细胞释放的ET进行比较。人类单细胞衍生巨噬细胞（HMDM）在接触不同炎症刺激后，在M1亲炎表型分化后产生MET。MET的释放可以通过显微镜可视化，使用绿色荧光核酸染色，不透过活细胞（例如，SYTOX绿色）。使用新鲜分离的原生巨噬细胞（如 HMDM）有利于模拟与潜在临床应用相关的体内炎症事件。该协议还可用于研究从人类单细胞细胞系（例如，THP-1）的MET释放后分化为巨噬细胞与磷酸酯或其他巨噬细胞系（例如，鼠巨噬细胞一样的J774A.1细胞）。

显示HMDM在细胞分化刺激中形态变化的图像如图1所示。M1 与暴露于 IFN® 和 LPS 的 HMDM 实验中产生的 M1 极化巨噬细胞显示了一个长而轴状的细胞形状，如图1（中间面板）中的黑色箭头所示。相比之下，M2极化巨噬细胞在暴露于IL-4444小时后的形态通常是圆形和扁平的，如图1（最右侧面板）中的黑色箭头所示。
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Watch this Scientific Journal Video about In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages at JoVE.com

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

这里介绍的是一种使用显微镜和荧光染色检测活细胞培养中巨噬细胞外陷阱 （MET） 生产的协议。此方案可进一步扩展，通过免疫荧光染色检查特定的 MET 蛋白标记物。

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 ...

中性粒细胞和其他免疫细胞释放细胞外陷阱在先天免疫中很重要，但也与炎症病理密切相关。虽然这些细胞在炎症反应中起着关键作用，但对巨噬细胞的这个过程了解得很少。该协议提供了一个主人类体外模型的巨噬细胞外陷阱或桅杆释放。它为我们研究这种结构在体内的潜在生理生长提供了一个模型。这项技术的主要优点是，从该协议的细胞是原细胞从人类的布衣制剂分离。无需启动步骤来区分单细胞到巨噬细胞，这是与其他单细胞细胞系，如THP-1细胞系的对比。该协议很容易适应其他免疫细胞，包括分离的嗜中性粒细胞和微胶质。巨噬细胞产生的细胞外陷阱非常脆弱，因此必须在步骤之间小心，以保持染色陷阱的完整性。在无菌条件下，通过加入完整的RPMI-1640培养基，将干扰素伽马添加到每毫升20毫微克的浓度和每毫升1微克的浓度中，准备M1吸油介质。通过将白细胞素 4 添加到完整的 RPMI-1640 培养基中，将 M2 启动介质添加到每毫升 20 毫微克的浓度中，准备 M2 启动介质。在无菌条件下，从含有HMDM的种子和培养组织培养板井中吸气培养介质。小心地加入每个井，一毫升无菌PBS预先加热到37摄氏度。拆下 PBS 缓冲液并再洗两次。向包含 HMDM 的每个井中添加一毫升的 M1 或 M2 注水介质。在细胞培养箱中存在5%的二氧化碳的情况下，在37摄氏度下孵育细胞48小时。在无菌条件下，将含有不同 MET 释放刺激器的培养基准备到完整的 RPMI-1640 介质。对于次氯酸刺激的实验，在具有HSS的猎鹰管中准备200微摩尔次氯酸，该管已预热至37摄氏度。极化治疗后，从每个井中吸出细胞介质，用一毫升的异同为一毫升的等同，用于PMA、TNFα和白细胞间8刺激，或HBSSs进行下氯酸刺激。在最后的洗涤步骤中去除 PBS 后，添加一毫升包含 PMA、TNF alpha 或白细胞素 8 的完整介质。在37摄氏度和5%的细胞培养箱中孵育细胞，用于TNF alpha刺激6小时，为PMA或白细胞间8刺激24小时。对于次氯酸的实验，在最后的洗涤步骤中去除HSSS后，加入一毫升新鲜准备的次氯酸。然后在37摄氏度下在细胞培养箱中存在5%的二氧化碳的情况下孵育细胞15分钟。之后，小心吸气细胞的上经剂，用一毫升的HSS等分液洗三次细胞。从最后的洗涤步骤中去除 HBSS 后，添加一毫升完整的 RPMI-1640 培养基。然后在37摄氏度下在细胞培养箱中存在5%的二氧化碳的情况下孵育细胞24小时。在HSSS中以40微摩尔的浓度准备SYTOX绿色染料。将染料的25微升直接添加到每个含有HMDM的井中。在黑暗中在室温下孵育细胞5分钟。然后将 HMDM 放在组织培养井中，放在倒置荧光显微镜的显微镜阶段进行成像。打开广谱荧光光源、亮场光源和带高分辨率彩色数码相机的倒置显微镜。将滤芯轮旋转至绿色荧光的二号位置，在 504 纳米时激发，尺寸在 523 纳米。使用 5 倍目标，用粗焦对焦对焦图像，然后将精细对焦旋钮聚焦在显微镜上，直到图像看起来清晰、清晰和聚焦。将显微镜切换到相机模式。启动关联的软件。单击"播放"按钮预览图像并调整显微镜上的精细对焦旋钮，直到图像在软件预览窗口中显示清晰、清晰和聚焦。单击"捕获"按钮。在软件中，单击"文件"。保存为所需的图像文件类型。在显微镜上，将滤光轮旋转到五号位置进行亮场成像，并重复捕捉过程以获取相应的亮场图像。此处显示了显示 HMDM 响应刺激的形态变化的亮场图像。M1极化巨噬细胞从HMDM实验暴露在干扰素伽马和脂质多糖显示一个拉长和主轴状的细胞形状。相比之下，HMDM暴露于白细胞间448小时后，M2极化巨噬细胞的形态通常是圆形和扁平的。通过SYTOX绿色活细胞荧光成像，将分化HMDM表型释放MEX的能力可视化。在没有任何亲炎刺激的情况下，从每个HMDM表型中孵育24小时的对照显示绿色染色非常有限。M1-HMDMs 接触次氯酸、PMA、白细胞素 8 或 TNF alpha，实现了 MET 的正染色（显示为绿色条纹）。细胞中明显的一些绿色染色的存在反映了膜完整性的丧失，这可能是由于细胞死亡而导致的，而细胞外陷阱释放。使用暴露于白细胞间4的M2-HMDM进行的实验表明，细胞中没有释放DNA，细胞外DNA的链或条纹的缺失就表明了这一点。然而，有一些细胞吸收绿色荧光染料与次氯酸和TNFα。在成像前避免直接亮光会过度暴露污渍，这一点很重要。细胞外DNA可以通过qPCR对细胞上因的核和线粒体DNA进行分析进行量化。与其他不朽的细胞系巨噬细胞储存相比，使用HMDM对桅杆结构及其在慢性炎症中的作用的进一步表征可能更具有临床相关性。

Research

JoVE Journal

Immunology and Infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

科研

免疫与感染

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

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

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 ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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 of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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 ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...