Name: bioparticle microarrays for chemotactic and molecular analysis of human neutrophil swarming in vitro
Uploaded: 2020-02-16T14:00:00
Description: Watch this Scientific Journal Video about Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro at JoVE.com

这项工作得到了威廉·劳里化学和生物分子工程系以及俄亥俄州立大学综合癌症中心的资助。本报告中提供的数据来自使用俄亥俄州立大学校园显微镜和成像设施提供的 Imaris x64（ver. 9.3.0 Bit 平面）处理的图像。该设施部分由国家癌症研究所P30 CA016058资助，贝塞斯达，医学博士。

作者感谢健康的志愿者谁亲切地献血。根据俄亥俄州立大学生物医学科学委员会审查的机构审查委员会（IRB）协议#2018H0268知情的志愿者同意后，获得血液标本。
1. 生物粒子微阵列的微观制造
<ol><li>使用标准光刻程序，生成主硅晶圆。<ol>
<li>使用计算机辅助设计 （CAD） 软件生成所需设计的证明，然后发送给照片蒙版制造商以生成镀铬照片蒙版。此处采用的设计是 4 mm x 4 mm ?...

<ol>
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我们开发了一个微冲压平台，以生成统一的生物粒子阵列，以刺激体外嗜中性粒细胞蜂群。我们的平台的体外性质使我们能够避免在体内蜂群实验产生的并发症，即分析由蜂群嗜中性粒细胞5释放的中介物的能力差。此外，体内模型通常在啮齿动物11，12，13，15，22，23</sup...

中性粒细胞，血流中最丰富的白细胞1，正在获得关注作为潜在的诊断和治疗目标2，3，因为他们可能涉及各种医疗条件，包括痛带4，败血症3，创伤5，6，癌症1，7，8和各种自身免疫性疾病5，9。中性粒细胞群是一个多阶段，严格监管的过程，其复杂性使它成为研究5，10，11特别有趣的焦点。在蜂拥而至期间，嗜中性粒细胞从周围的健康组织5、10、11分离出炎症部位。正确调节嗜中性粒细胞蜂群对于促进伤口愈合并最终解决炎症决议5，12是必要的。中性粒细胞群主要研究啮齿动物12，13，14，15和斑马鱼10，11，12，15模型在体内。然而，这些体内动物模型的性质引起限制5。例如，在蜂群中由嗜中性粒细胞释放的介质不容易被分析5。此外，在体内有许多潜在的中介，因此体内实验必须引入基因缺陷来抑制细胞生产和/或相互作用，以调查该中介在给定过程中的作用13。体外实验通过启用中性粒细胞观察来规避这种并发症，而无需额外的细胞上下文。此外，描述人类中性粒细胞协调迁移的研究也有限。在体外蜂群平台上，可以直接分析人类嗜中性粒细胞。体外蜂群平台可以通过提供机会来填补体内研究的局限性所留下的空白，从而扩大从体内研究中获得的知识。
为了满足对模仿体内嗜中性粒细胞群的体外平台的需求，我们开发了一个微冲压平台，使我们能够以空间控制的方式对生物粒子微阵列进行阵列，以刺激嗜中性粒细胞蜂群。我们分两步在玻璃玻片上生成生物粒子微阵列。首先，我们使用微冲压来生成阳离子聚电解质 （CP） 位微阵列。其次，我们添加通过静电相互作用粘附在CP点的生物粒子溶液。通过首先对CP层进行图案化，我们可以有选择地对带负电荷的生物粒子进行模式化，以生成所需的中性粒细胞蜂群模式。带正电荷的层通过剧烈的洗涤步骤将生物粒子从玻璃滑道上没有CP的区域移除。 此外，这里使用的CP，丙烯酰胺和四元阳离子单体的共聚物，具有生物相容性，因此不会引起中性粒细胞的反应。它具有非常高的表面电荷，使微米大小的生物颗粒固定到玻璃滑动，从而阻止嗜中性粒从玻璃滑轨上的图案位置去除颗粒。这导致生物粒子簇排列在微阵列中。当我们在微阵列中加入嗜中性粒细胞时，它们在生物粒子簇周围形成了稳定的群。通过跟踪中性粒细胞迁移，我们发现成群的中性粒细胞会主动向生物粒子簇迁移。此外，我们利用这个平台分析中性粒细胞在蜂拥过程中释放的某些介质。我们发现16个调解员在蜂拥而至时表达不同。随着时间的推移，它们的浓度遵循三个总趋势：增加、减少或峰值。我们的体外中性粒细胞蜂群平台有助于分析空间控制的人类嗜中性粒细胞蜂群，以及收集和分析由嗜中性粒细胞蜂群释放的介质。在以前的出版物中，我们证明了患有某些医疗条件（创伤、自身免疫性疾病和败血症）的患者，其功能与健康捐赠者不同。在未来的研究中，我们的平台可用于分析各种患者群体中的中性粒细胞功能。该平台可以定量分析中性粒细胞群中涉及的复杂协调。可以进行其他研究，以深入了解特定患者群体的嗜中性粒细胞功能或对感兴趣的病原体的嗜中性粒细胞反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#34;The Big Easy&#34; EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18001</td>
			<td>Magnet to use with neutrophil isolation kit</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Okolab</td>
			<td>777057437 / 77057343</td>
			<td>Okolab cage incubator for temperature and CO2 control</td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Isolation Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17957</td>
			<td>Kit for immunomagnetic negative selection of human neutrophils</td>
		</tr>
		<tr>
			<td>Eclipse Ti2</td>
			<td>Nikon Instruments</td>
			<td>MEA54010 / MEF55037</td>
			<td>Inverted research microscope</td>
		</tr>
		<tr>
			<td>Escherichia coli (K-12 strain) BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag&#174; array</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 8-mm biopsy punch</td>
			<td>Sigma Aldrich</td>
			<td>Z708925</td>
			<td>To cut PDMS stamps</td>
		</tr>
		<tr>
			<td>HetaSep</td>
			<td>STEMCELL Technologies</td>
			<td>7906</td>
			<td>Erythrocyte aggregation agent for separating buffy coat from red blood cells in fresh human blood</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td>Nucleus fluorescent stain</td>
		</tr>
		<tr>
			<td>Human L1000 Array</td>
			<td>Raybiotech Inc.</td>
			<td>AAH-BLG-1000-4</td>
			<td>High density array to detect 1000 human proteins</td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Sigma Aldrich</td>
			<td>A5843</td>
			<td>Low endotoxin HSA, to prepare 2 % solutions in IMDM for isolated neutrophils</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbeccos&#39; Medium (IMDM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td>To resuspend isolated neutrophils</td>
		</tr>
		<tr>
			<td>K2-EDTA tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-657-32</td>
			<td>Tubes for blood collection</td>
		</tr>
		<tr>
			<td>Low Reflective Chrome Photomask</td>
			<td>Front Range Photomask</td>
			<td>N/A</td>
			<td>Dimensions 5&#34; x 5&#34; x 0.09&#34; (L x W x D)</td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Perkin Elmer</td>
			<td>ASCNGX00</td>
			<td>Fluorescence reader of protein patterned microdomains</td>
		</tr>
		<tr>
			<td>Microscopy Image Analsysis Software - Imaris</td>
			<td>Bitplane</td>
			<td>9.3.0</td>
			<td>Software for automatic cell tracking analysis</td>
		</tr>
		<tr>
			<td>NiS Elements Advanced Research Software Package</td>
			<td>Nikon Instruments</td>
			<td>MQS31100</td>
			<td>Software for automatic live cell imaging and swarm size calculation</td>
		</tr>
		<tr>
			<td>Poly-L-lysine fluorescein isothiocyanate (PLL-FITC)</td>
			<td>Sigma Aldrich</td>
			<td>P3069-10MG</td>
			<td>30,000 - 70,000 MW PLL labeled with FITC, used to fluorescently label CP solution</td>
		</tr>
		<tr>
			<td>SecureSeal 8-well Imaging Spacer</td>
			<td>Grace Bio-Labs</td>
			<td>654008</td>
			<td>8-well, 9-mm diameter, adhesive imaging spacer</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>590</td>
			<td>Silicon 100 mm N/P (100) 0- 100 ohm-cm 500 &#956;m SSP test</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell</td>
			<td>WS-650MZ-23NPPB</td>
			<td>Used to spincoat a 40-&#181;m layer of photoresist onto silicon wafer</td>
		</tr>
		<tr>
			<td>SU-8 2025</td>
			<td>MicroChem</td>
			<td>2025</td>
			<td>Negative photoresist to make silicon master wafer</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Photoresist developer. Remove non-crosslinked SU-8 2025 from silicon wafer</td>
		</tr>
		<tr>
			<td>Sylgard 184 (polydimethylsiloxane, PDMS)</td>
			<td>Dow</td>
			<td>1673921</td>
			<td>2-part silicone elastomer kit for making microstamps and PDMS wells</td>
		</tr>
		<tr>
			<td>UV Exposure Masking System</td>
			<td>Klo&#233;</td>
			<td>UV-KUB 2</td>
			<td>Used to crosslink photoresist on silicon wafer through chrome mask with UV light</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1287303</td>
			<td>High quality water to dilute bioparticles</td>
		</tr>
		<tr>
			<td>Zetag 8185</td>
			<td>BASF</td>
			<td>8185</td>
			<td>Cationic polyelectrolyte (CP), powder, Copolymer of acrylamide and quaternized cationic monomer, forms &#34;inking solution&#34; for microstamping when dissolved in water</td>
		</tr>
		<tr>
			<td>Zymosan A S. cerevisiae BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>Z2843</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag array</td>
		</tr>
	</tbody>
</table>

bioparticle microarrays for chemotactic and molecular analysis of human neutrophil swarming in vitro

中性粒细胞群是一个合作的过程，通过它，嗜中性粒细胞封闭一个感染部位，并促进组织重组。在动物模型中，在动物模型中对蜂拥性进行了经典的研究，显示了细胞迁移的特征模式。然而，在体内模型有几个限制，包括细胞间介质难以访问和分析，以及无法直接分析人类嗜中性粒细胞。由于这些限制，需要一个体外平台，研究蜂拥与人类嗜中性粒细胞，并提供容易访问在蜂群生成分子信号。在这里，一个多步骤的微冲过程用于生成生物粒子微阵列，通过模仿体内感染来刺激蜂群。生物粒子微阵列以可控、稳定的方式诱导嗜中性粒细胞成群。在微阵列上，嗜中性粒细胞在生物粒子簇周围增加速度并形成稳定的群。此外，对嗜中性粒细胞产生的上清液进行了分析，发现16种蛋白质在蜂群过程中表达得不同。这个体外蜂群平台有助于以可重复的空间控制方式直接分析中性粒细胞迁移和蛋白质释放。

当中性粒细胞添加到生物粒子微阵列中时，接触生物粒子簇的嗜中性粒细胞被激活并启动聚集反应。生物粒子微阵列使用延时荧光显微镜进行验证，以跟踪中性粒细胞向生物粒子簇的迁移（视频S1）。单个嗜中性粒细胞核在向生物粒子簇迁移时被跟踪。当嗜中性粒细胞到达生物粒子簇时，其核与星团中的其他核重叠。因此，无法使用此方法准确跟踪群集中的嗜中性粒细胞。日莫桑和<em...

Watch this Scientific Journal Video about Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro at JoVE.com

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

该协议生成生物粒子微阵列，提供空间控制的嗜中性粒细胞群。它便于接触嗜中性粒细胞在迁移过程中释放的介质，并允许定量成像分析。

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

该协议是重要的，因为它允许对中性粒细胞群进行体外分析，从而克服了我们目前无法进行实验的许多限制。该技术的主要优点是，它提供了容易访问分泌的细胞因子和脂质介质，中性粒细胞在蜂拥期间释放，并允许定量成像分析。当我们将这项技术应用于嗜中性粒细胞时，它可以扩展以分析其他白细胞的迁移，如单细胞和T细胞。首先，制备每毫升1.6毫克的阳离子多电子光石或CP溶液，将适当数量的CP粉末加入水中，并在搅拌板上混合过夜，或直到所有固体溶解。如果需要，可以通过添加标有 FITC 的多 L-lysine 来使溶液成为荧光。使用标准光刻程序生成主硅晶圆。此处使用的设计是一个四到四毫升的矩形阵列，直径为 30 微米填充的圆圈，具有 150 微米的中心到中心间距，但可以根据需要修改，以适合不同的应用。将一层 40 微米厚的负光处理层旋转到硅晶圆上。然后在65摄氏度下烘烤晶圆5分钟。和95摄氏度10分钟。通过镀铬光面罩将晶圆暴露在紫外光照下，每厘米方为 150 至 160 毫焦耳。在65摄氏度下再次烘烤晶圆5分钟，95摄氏度烘烤10分钟。然后潜入光师开发人员 10 分钟。用异丙醇冲洗。该模式现在应在晶圆上可见。接下来，彻底混合聚二甲基硅氧烷（PDMS）及其固化剂的10比1的比例。将未处理的混合物倒在培养皿的主晶圆上。真空处理未加热的 PDMS 混合物，直到不存在气泡。然后在65摄氏度过夜治愈。第二天，使用手术刀切割晶圆的图案部分的外部。然后慢慢拆下固化的 PDMS 板，将其放置在带图案的朝上清洁的切割板上。用 8 毫米活检冲孔从 PDMS 板中打出单独的邮票，并面朝下将每个邮票放在胶带上以清除碎屑。随着这些邮票朝上，每个邮票的底面与100微升的预先准备的CP解决方案，确保没有气泡形成之间的解决方案和邮票。然后将图章倒置到 CP 溶液的一层上，并在一小时后将其删除。将每个湿印压面向下涂抹在干净的玻璃滑梯上六到八次，以去除多余的液体。然后真空处理邮票一到两分钟。在干净的玻璃滑梯上粘上八孔、八毫米直径的成像空间，作为邮票放置的指南。将图章面朝下放在成像空间器每个井中央的玻璃幻灯片上。然后在每个邮票上放置一个 5.6 克的平衡重量，并留出 10 分钟进行冲压。从幻灯片上取下重量和图章，让 CP 层在室温下干燥 24 小时，然后再添加生物颗粒。如果 CP 带有 FITC 标记，则使用荧光显微镜检查冲压的有效性。将空白 PDMS 板切割到成像分张器的大小，并使用 8 毫米活检冲孔在 PDMS 中创建与消热器孔对齐的孔。然后将 PDMS 板粘附在玻璃幻灯片上。解冻生物粒子溶液，稀释至每毫升500微克进行注射。在玻璃滑梯上的每个 PDMS 井中加入 100 微升的生物颗粒溶液，然后摇动幻灯片 30 分钟。用水彻底冲洗水，并用荧光显微镜检查幻灯片上的图案。生物粒子微粒可以在4摄氏度的无尘环境中储存长达三个月。在 IMDM 中，每毫升 7.5 倍至第五毫升细胞用 0.4% 的人血清白蛋白重新产生细胞，以准备细胞。将悬浮液的100微升添加到含有生物粒子微孔的PDMS井中，确保细胞悬浮液在PDMS顶部凸升，且不含任何气泡。用直径为 12 毫米的盖滑封住井，然后用钳子轻轻按下，使多余的细胞悬浮液逃到井的边缘，然后使用纸巾去除多余的。要对细胞进行成像，在装有37摄氏度、5%二氧化碳和90%相对湿度的显微镜的活细胞成像站的活细胞成像站的细胞上加载微粒阵列。使用时差、荧光和亮场显微镜，在 405 纳米、594 纳米和明亮场下，每 10 秒以 10 倍放大率记录图像。在37摄氏度和5%的二氧化碳下用生物粒子在井中孵育嗜中性粒细胞3小时，在所需时间点采集样本。使用明亮的场显微镜验证在微阵列上形成成群的。收集单个井的上清液的整个体积，并加载到0.45微米的离心过滤管中，确保分析三重的每个时间点。在190倍的G和20摄氏度下离心管5分钟，并收集过滤体积。然后将样品储存在零下80摄氏度，直到处理时间。尝试此协议时，请与烟罩中的光处理师和开发人员合作。在从人类献血者那里吸血之前，请制定 IRB 批准的方案。请记住，从人类血液中衍生的材料是 BSL2。当嗜中性粒细胞被添加到生物粒子微阵列中时，接触生物粒子簇的嗜中性粒细胞被激活并启动蜂群反应。利用延时荧光显微镜验证了生物粒子微量阵列，以跟踪嗜中性粒细胞向生物粒子簇的迁移。在30至60分钟的暴露后，每个聚类周围形成稳定的嗜中性粒细胞群。相比之下，在没有生物粒子簇的情况下，嗜中性粒细胞不会表现出集体迁移。染色中性粒细胞核的荧光强度用于确定生物粒子团周围的平均群大小约为1490微米平方。在没有聚类的情况下，给定感兴趣区域的荧光会随着时间的推移而保持不变。嗜中性粒细胞迁移的轨迹表明，当存在微粒组时，嗜中性粒细胞聚集在生物粒子簇上，但在控制系统中未观察到汇合。测量了蜂拥和非活化中性粒细胞的速度，发现速度分布有统计学上显著差异。成群的嗜中性粒细胞的平均速度为每分钟20.6微米，而控制嗜中性粒细胞的平均速度为每分钟2微米。随着时间的推移，对嗜中性粒细胞在蜂拥而至期间释放的16种蛋白质的浓度进行了分析。10种蛋白质的浓度增加。两个减少。其余四个在蜂拥而至的第一个小时内有所增加，但之后减少了。执行此过程时，请确保所有内容清洁，并且您的邮票正确干燥，因为这些对于细菌颗粒的成功图案至关重要。这项技术的发展将使研究人员能够探索嗜中性粒细胞群领域的新问题，包括自由调停器和细胞外囊泡如何参与中性粒细胞间交流。

Research

JoVE Journal

Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk Film Culture System for in vitro Analysis and Biomaterial Design

丝绸电影文化系统在体外分析和生物材料设计

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

生产自体富血小板血浆以促进体外人成纤维细胞扩增

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...