Name: simultaneous study of the recruitment of monocyte subpopulations under flow in vitro
Uploaded: 2018-11-26T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro at JoVE.com

我们感谢保罗·布拉德菲尔德博士的手稿阅读和反馈。a. s. 得到了朱尔斯·索恩爵士慈善海外信托委员会的财政支持。

人类材料是在自愿捐助者知情同意的情况下, 根据瑞士临床研究道德委员会使用的。
1. 人脐静脉内皮细胞的分离和冷冻 (huvec)
<ol><li>在 ph 为7.4 的情况下, 在 ph 为7.4 的情况下, 在 ph 值7.4 下, 在 t75 瓶中加入5毫升的涂层溶液, 在37°c 下进行30分钟的涂装液。</li>
	<li>用 pbs 清洁绳子, 用无菌压缩器擦拭, 并将其放入20厘米的无菌培养皿中。用无菌剪刀剪断绳子的两端。</li>
	<li>识别?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+monocytes+consist+of+two+principal+subsets+with+distinct+migratory+properties.">Blood monocytes consist of two principal subsets with distinct migratory properties.</a> Immunity. 19 (1), 71-82 (2003).</li><li>Chamorro, 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=In+vitro+differentiation+of+porcine+blood+CD163&#8722;+and+CD163++monocytes+into+functional+dendritic+cells.">In vitro differentiation of porcine blood CD163&#8722; and CD163+ monocytes into functional dendritic cells.</a> Immunobiology. 209 (1-2), 57-65 (2004).</li><li>Passlick, B., Flieger, D., Ziegler-Heitbrock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+monocyte+subpopulation+in+human+peripheral+blood.">Identification and characterization of a novel monocyte subpopulation in human peripheral blood.</a> Blood. 74 (7), (1989).</li><li>Venneri, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotion+of+monocytes+on+endothelium+is+a+critical+step+during+extravasation.">Locomotion of monocytes on endothelium is a critical step during extravasation.</a> Nature Immunology. 5 (4), 393-400 (2004).</li><li>Luu, N. T., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+different+steps+during+neutrophil+migration+through+cultured+endothelial+monolayers+treated+with+tumour+necrosis+factor-alpha.">Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-alpha.</a> Journal Vascular Research. 36 (6), 477-485 (1999).</li><li>ibidi GmbH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Shear Stress and Shear Rates for ibidi &#181;-Slides - Based on Numerical Calculations. , (2014).</li><li>Yang, L., Froio, R. M., Sciuto, T. E., Dvorak, A. M., Alon, R., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICAM-1+regulates+neutrophil+adhesion+and+transcellular+migration+of+TNF-alpha-activated+vascular+endothelium+under+flow.">ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow.</a> Blood. 106 (2), 584-592 (2005).</li><li>Yang, C. -. R., Hsieh, S. -. L., Ho, F. -. M., Lin, W. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoy+receptor+3+increases+monocyte+adhesion+to+endothelial+cells+via+NF-kappa+B-dependent+up-regulation+of+intercellular+adhesion+molecule-1,+VCAM-1,+and+IL-8+expression.">Decoy receptor 3 increases monocyte adhesion to endothelial cells via NF-kappa B-dependent up-regulation of intercellular adhesion molecule-1, VCAM-1, and IL-8 expression.</a> Journal of Immunology. 174 (3), 1647-1656 (2005).</li><li>Wong, D., Dorovini-Zis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+vascular+cell+adhesion+molecule-1+(VCAM-1)+by+human+brain+microvessel+endothelial+cells+in+primary+culture.">Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture.</a> Microvascular Research. 49 (3), 325-339 (1995).</li><li>Bradfield, P. F., Nourshargh, S., Aurrand-Lions, M., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAM+family+and+related+proteins+in+leukocyte+migration+(Vestweber+series).">JAM family and related proteins in leukocyte migration (Vestweber series).</a> Arteriosclerosis Thrombosis and Vascular Biology. 27 (10), 2104-2112 (2007).</li><li>Bradfield, P. 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=Divergent+JAM-C+Expression+Accelerates+Monocyte-Derived+Cell+Exit+from+Atherosclerotic+Plaques.">Divergent JAM-C Expression Accelerates Monocyte-Derived Cell Exit from Atherosclerotic Plaques.</a> PLoS One. 11 (7), e0159679 (2016).</li></ol>

在这里, 我们报告了一个方法详细介绍了单核细胞亚群如何通过发炎的内皮单层迁移的研究。讨论的方法采用共聚焦显微镜代替相对比显微镜, 也用于研究流3、11、19 下单核细胞的招募。使用共聚焦显微镜进行延时成像的一个主要优点是能够明确区分单核细胞的迁移和强粘附。虽然基于相对比显微镜的方法也很稳健, 但它需要专...

单核细胞是先天免疫的吞噬成分, 对于对抗病原体、清理受损组织、血管生成以及包括癌症在内的许多疾病的病理生理学至关重要..单核细胞是由异质亚群组成的骨髓细胞, 在血液中循环, 但可以通过特定的分子机制被招募到外周组织的炎症部位。与一般白细胞一样, 单核细胞的招募级联涉及不同的步骤, 包括捕获、滚动、爬行、逮捕、内皮迁移 (转移) 和通过血管壁 (基底膜和壁画) 的迁移单元格)4。这些步骤主要涉及炎症引起的内皮腔表面的分子, 如选择、糖蛋白配体、趋化因子、细胞间和结合粘附分子, 以及它们对白细胞的受体, 如选择性配体和整合。通过内皮细胞连接 (脱细胞后) 或通过内皮细胞体 (跨细胞) 的贩运途径可被白细胞用于跨越内皮屏障5。虽然历史上有记录表明单核细胞通过跨细胞途径迁移, 但由于单核细胞不再被视为同质细胞群, 它们迁移途径中的潜在差异已被提出。现在越来越清楚的是, 单核细胞多样性可以通过它们的每一个差异和共性来定义, 就它们独特的外渗级联 3,6。因此, 为了明确区分单核细胞亚群, 在招募过程中, 将这些不同亚群的行为形象化和表型是至关重要的。
将人、猪、大鼠和小鼠的单核细胞细分为表型亚群, 具有一定的归因功能和特定的迁移行为7,8,9。例如, 在人类中, 单核细胞可以根据其表面表达的 cd14 (细菌脂多糖的核心受体) 和 cd16 (fc-γ受体 iii) 分为三个子集。人类单核细胞亚群包括经典 cd14+cd16-、中间 cd14+cd16+和非经典 cd14 暗淡 cd16+细胞6,9。经典 cd14+cd16-单核细胞主要具有炎症性, 而 cd16 + 单核细胞池被共同发现为 tie2表达和血管生成功能10。持续, 内皮细胞刺激与炎症细胞因子, 如人类肿瘤坏死因子 (tnf) α或白细胞介素 (il-1)beta (常规炎症) 足以引发经典 cd14+cd16 的完全招募-单核细胞然而, 血管内皮生长因子 (vegf) a 和 tnfα (血管生成因子引起的炎症) 同时作用, 是引发 cd16 + 单核细胞血管生成池 3的迁移.从历史上看, 传统的跨井系统在静态条件下, 平行板流动室和μ-滑动流室已被用来定量分析在体外一次招募一个白细胞群 11 ,12,13。虽然这些方案已经得到验证, 一个更强大的方法, 允许同时分析多个单核细胞亚群将被认为更有见地。这种方法必须考虑到多个相互作用和每个群体的不同频率, 并提供机械地理解定义每个单核细胞的招募级联的相似性和特殊性子集。
本文提出了一种基于流动过程中单核细胞招募延时成像的方法, 该方法可以利用共聚焦显微镜同时研究不同单核细胞亚群的迁移级联。该方法结合了某些关键特征, 模仿内皮细胞炎症, 以及在毛细血管后静脉循环单核细胞的血流动力学, 白细胞在体内招募的主要位置。该方法使用人脐静脉内皮细胞 (huvec), 这是通过与人脐带分离的既定协议产生的。这种临床资源的优点是很容易作为生物副产品获得, 同时也提供了一个合理的内皮细胞产量, 可以从脐静脉分离。我们还使用荧光染料和免疫荧光来区分不同的细胞成分, 并使用共聚焦显微镜明确定义单核细胞定位 (腔内与烧黑细胞) 随着时间的推移。本文所介绍的协议是为了同时测量单核细胞亚群的迁移水平而制定的。此外, 应当指出, 这种方法可以通过使用不同的生物标志物和标签, 扩大到研究其他白细胞亚群和招募过程。

<table>
	<tbody>
		<tr>
			<td>Tissue Culture Flasks 75 cm2</td>
			<td>TPP</td>
			<td>90076</td>
			<td>Routine culture of isolated HUVEC</td>
		</tr>
		<tr>
			<td>&#181;-Slide VI 0.4</td>
			<td>IBIDI</td>
			<td>80606</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes 15 mL</td>
			<td>TPP</td>
			<td>191015</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes 50 mL</td>
			<td>TPP</td>
			<td>191050</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen G</td>
			<td>Biochrom</td>
			<td>L 7213</td>
			<td>For coating of cell culture flasks</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>1393</td>
			<td>For coating of cell culture flasks</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (without MgCl2 and CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (with MgCl2 and CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Way Stopcocks</td>
			<td>BIO-RAD</td>
			<td>7328103</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin 10000 u/ml streptomycine 10000 ug/ml fungizone 25 ug/ml</td>
			<td>AMIMED</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1</td>
			<td>Worthington</td>
			<td>LS004216</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199 1X avec Earle&#39;s salts, L-Glutamine, 25 mM Hepes</td>
			<td>GIBCO</td>
			<td>22340020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin Fraction V</td>
			<td>ThermoFisher</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Supplement, 150mg</td>
			<td>Millipore</td>
			<td>02-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium</td>
			<td>Sigma-Aldrich</td>
			<td>H3149RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H6909</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A 4544</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate C10H14N2Na2O8 &#183; 2H2O</td>
			<td>APPLICHEM</td>
			<td>A2937.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD144 (VE-Cadherin), human recombinant clone: REA199, FITC</td>
			<td>Miltenyi Biotech</td>
			<td>130-100-713</td>
			<td>AB_2655150</td>
		</tr>
		<tr>
			<td>CD31-PE antibody, human recombinant clone: REA730, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-110-807</td>
			<td>AB_2657280</td>
		</tr>
		<tr>
			<td>Anti-Podoplanin-APC, human recombinantclone: REA446, APC</td>
			<td>Miltenyi Biotech</td>
			<td>130-107-016</td>
			<td>AB_2653263</td>
		</tr>
		<tr>
			<td>BD Accuri C6 Plus</td>
			<td>BD Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide I Luer</td>
			<td>IBIDI</td>
			<td>80176</td>
			<td></td>
		</tr>
		<tr>
			<td>CMFDA (5-chloromethylfluorescein diacetate)</td>
			<td>ThermoFisher</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TNF&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGFA</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>NE-1000 Programmable Syringe Pump</td>
			<td>KF Technology</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#160;Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD14-PE, human recombinant clone: REA599, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-110-519</td>
			<td>AB_2655051</td>
		</tr>
		<tr>
			<td>Pan Monocyte Isolation Kit, human</td>
			<td>Miltenyi Biotech</td>
			<td>130-096-537</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD16-PE, human recombinant clone: REA423, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-106-762</td>
			<td>AB_2655403</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotech</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>ThermoFisher</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>IBIDI</td>
			<td>10841</td>
			<td></td>
		</tr>
		<tr>
			<td>Elbow Luer Connector</td>
			<td>IBIDI</td>
			<td>10802</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer Lock Coupler</td>
			<td>IBIDI</td>
			<td>10823</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock Connector Female</td>
			<td>IBIDI</td>
			<td>10825</td>
			<td></td>
		</tr>
		<tr>
			<td>In-line Luer Injection Port</td>
			<td>IBIDI</td>
			<td>10820</td>
			<td></td>
		</tr>
		<tr>
			<td>Ar1 confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40X objective</td>
			<td>Nikon</td>
			<td>40x 0.6 CFI ELWD S Plane Fluor WD:3.6-2.8mm correction 0-2mm</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous study of the recruitment of monocyte subpopulations under flow in vitro

在组织损伤、肿瘤发育和自身免疫性疾病的过程中, 从血液中招募单核细胞到靶向外周组织对炎症过程至关重要。这是通过捕获过程从自由流动到腔表面的激活内皮细胞, 其次是他们的粘附和内皮迁移 (转移) 到底层受影响的组织。然而, 支持优先和与上下文相关的单核细胞亚群招募的机制仍未得到充分理解。因此, 我们开发了一种方法, 允许在流量下同时对不同单核细胞亚群的招募进行可视化和测量。这种基于延时共聚焦成像的方法允许在粘附体和迁移单核细胞之间进行明确的区分。在这里, 我们描述了如何使用这种方法可以同时研究招募的亲血管生成和非血管生成单核细胞在体外。此外, 这种方法还可以推广到研究最多三个单核细胞种群的不同招募步骤。

tnfα诱导 huvec 活化状态的测定
炎症细胞因子 tnfα的生物活性可根据冻融循环的批次和重复而变化。tnfα治疗对 huvec 的活化状态进行检查是非常重要的。这可以通过平行染色的一些融合 huvec 样品进行炎症诱导选择, icam-1 和 vcam-115,16,17。一种更简便的方法来检查...

Watch this Scientific Journal Video about Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro at JoVE.com

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

在这里, 我们提出了一个综合协议, 测量单核细胞亚群贩运在流在体外使用特定的表面标记和共聚焦荧光显微镜。该协议可用于探索顺序招募步骤, 以及使用其他特定的表面标记来描述其他白细胞亚型。

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

因为这种方法可以帮助回答炎症和免疫学领域的关键问题。例如，白细胞粘附和跨内皮迁移的分子机制是什么？这种技术的主要优点是，它允许无限使用之间的异化和强添加单核细胞到内皮单层之间的无限使用。虽然这种方法提供了有关在流中招募单细胞的信息，但它也可以适应造血系统的其他细胞，如T细胞、B细胞或亚细胞的衍生物。洗涤后，用30微升37摄氏度M199介质标记人体脐静脉内皮细胞或HUVEC，辅以1微摩尔CMFDA，在37摄氏度和5%的二氧化碳下进行10分钟。在孵育结束时，用150微升完整M199培养的培养液清洗细胞，并在150微升新鲜完整M199培养的培养中再孵育30分钟。然后用150微升的完整的M199介质，包含适当的实验补充剂，并返回幻灯片室培养器6小时。对于人类PAN单细胞分离，稀释PBS中新鲜采集的血液样本，以一比一的比例补充1毫摩尔EDTA，并小心地将20ml血液溶液分层到20ml密度梯度介质上，通过离心进行密度梯度分离。将中外周外体血细胞血小板层收集到含有 40 ml PBS 的新的 50 ml 管中，并辅以 1 毫摩尔 EDTA，并在计数后将最终体积与额外的 PBS-EDTA 一起放入 50 ml，然后根据制造商的说明使用 PAN 单核细胞分离试剂盒分离单核细胞。在M199介质中三次清洗细胞，辅以0.5%牛血清白蛋白或BSA，以消除EDTA的任何痕迹。单细胞分离的质量对于确保强大的迁移和粘附结果至关重要。因此，必须严格遵守制造商的隔离建议。在新鲜M199介质中，以每毫升6倍至6个单核细胞浓度重新加入颗粒，并辅以0.5%BSA，将细胞分成每微离心管200微升等分法。然后将细胞在37摄氏度下放置20分钟，然后注射到流室。要准备用于分析的流体系统，请先将公 luer 连接器插入 8 厘米长 3 毫米厚的硅胶管的一端，然后将另一端连接到内联 luer 喷射装置。然后将 luer 连接器连接到一块 40 厘米长、3 毫米厚的硅胶管的一端。接下来，将 20 mm 注射器插入一端 1 米长 3 毫米厚的硅胶管，并将公用 luer 连接器插入另一端。然后将两个公 luer 连接器插入母 luer 锁连接器，并将硅胶管的自由端放入 37 摄氏度 M199 介质的储液罐中，并辅以 0.5%BSA。缩回柱塞，用流量缓冲液填充油管，并固定注射器泵上的注射器。然后将泵设置为提取模式并指定流量。要连接滑动室，请将硅胶管夹在母凸锁连接器周围，然后断开两个 luer 连接器与连接器的母连接器，以便将其连接到滑轨的储液罐。气泡不仅干扰图像质量，而且会对细胞产生毛细管纯粹的压力，导致细胞死亡。为避免气泡，在将凸流器插入储液罐之前，请确认油管的正确夹紧。然后拆下夹子以确认连接未泄漏，然后将幻灯片置于显微镜下。为对流动下的单细胞招募进行成像，在室温下将5微升面粉结合抗CD16抗体和适当的核染料加入每个等分单核细胞，在室温下孵育10分钟，并采集具有短暂离心的细胞。将颗粒重新注入 250 微升流量缓冲液中，然后启动注射器泵。将一个单核细胞悬浮液的30微升添加到幻灯片的一个腔室，并在共合显微镜上选择40倍的镜面。激活适当的荧光激光器，并使用标有单细胞的腔室设置共合体显微镜的采集参数"下一步"，选择三个视场在半厘米半径内进行多位置共声成像，将 Z 堆栈设置为 10 到 12 微米范围，并且每一分钟运行一次时间推移采集。经过三分钟的成像，通过内联 luer 注射口注入 200 微升单核细胞，并开始成像细胞相互作用。至少 30 分钟后，停止成像和流量并夹紧油管，以便将其与幻灯片断开。要通过受刺激的HUVECs确定细胞的迁移速率，请打开图像J并计算每个字段中粘附单细胞的总数，以确定每平方毫米的细胞计数。然后计算在内皮细胞下方的基底平面中存在的异化单核的数量，如细胞核周围黑洞区的存在所识别。一个简单的方法来检查HUVECs的激活状态刺激后是可视化其在炎症压力下的伸长。时间推移共物成像允许在可评估其表型的平面上对单细胞进行可视化。迁移的细胞在从平面上消失并重新出现在基底平面之前，会移动到细胞间细胞到细胞的交汇点。随着时间的推移，单细胞招募的定量证实，单细胞粘附后跟转位。当内皮细胞仅用TNF alpha刺激时，CD16阳性单细胞的迁移率较低，但当内皮细胞同时使用TNFα和血管内皮生长因子或维格法刺激时，迁移率会上升。与CD14阳性单细胞相比，使用TNF alpha和TNF alpha加VEGFA的HUVEC刺激诱导CD14阴性T、B和NK细胞的依从性增加。此外，HUVEC刺激只诱导单细胞种群的迁移，因为T、B和NK细胞在TNF alpha或TNF alpha加上VEGFA刺激条件下不会迁移。要进行最佳的单核细胞招募测定，重要的是您提前计划实验并在同一天进行。当您净化细胞时，请确保您的显微镜在37度，这样在实验中不会将细胞转移到不同的温度。为了学习这个程序，可以执行所有的方法，如HUVEC细胞因子和化疗表达的特征分析，以及化疗研究，以回答有关分子机制的附加问题，维持特定单细胞群体迁移和粘附。

Research

JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

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 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

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

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 ...

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 ...