Name: microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood
Uploaded: 2014-06-03T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

来自美国国立卫生研究院支持（授予GM092804，DE019938）和烧伤的Shriners医院。

1，微流控设备制造<ol><li>使用标准光刻技术，制作了1000级洁净室的母模晶片。图案的第3μm的薄环氧系负光刻胶层，根据来自制造商的说明来定义迁移通道。图案的第二50微米厚的层来定义小区负载和趋化因子的腔室。 </li><li>使用图案化的晶片投聚二甲基硅氧烷（PDMS）的设备。大力混合的PDMS（20克）与引发剂（2克），使用塑料叉子在大型塑料托盘称重5分钟。 </li><li>小心倒出的PDMS模具上。 </li>...

<ol>
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在这项工作中，我们开发了一个微流体平台，从血液滴（2微升）测量嗜中性粒细胞的趋化性。红细胞从积极的中性粒细胞迁移的芯片上的机械过滤规避了繁琐的细胞分离方法如密度梯度10，正选择11或阴性选择12，这很容易通过激活嗜中性粒细胞引入伪像。红细胞的机械过滤区分我们的技术与其他微流控芯片，用于探测中性粒细胞迁移。例如，从本实验室13和以往?...

<html>嗜中性粒细胞贩卖起着决定的许多炎症性疾病，包括动脉粥样硬化1，细菌感染或败血症2的进度和分辨率的关键作用，以及烧伤3。对于健康和疾病状况做出的巨大贡献，中性粒细胞计数的标准血液分析通常被认为在临床和科研实验室的一部分。然而，尽管是一种最普遍的测试，中性粒细胞计数在感染和败血症的诊断价值已频频质疑4。例如，在烧伤病人的中性粒细胞的一项研究发现，嗜中性粒细胞计数和中性粒细胞迁移的功能不相关;标志着单独的中性粒细胞计数是不是免疫状态3的准确指标。虽然越来越多的难以衡量，中性粒细胞功能的能力提出了更有价值的各种条件。 T“&gt;重要的是，许多嗜中性粒细胞缺陷是短暂的，不会被永久的遗传缺陷，已经在很大程度上忽略了诊所，直到最近的区分触发。在烧伤的情况下，中性粒细胞迁移能的过程中被监控病人的治疗，炎症状态或感染3的指标。目前在实验室（Boyden小室，邓恩室，微量法）使用传统迁移实验不能转换成临床上，因为它们需要大量的血液和繁琐费时嗜中性粒细胞分离技术（ 表1）。这些测定也不能用来监测在小的实验室动物，如小鼠的嗜中性粒细胞趋化性的瞬时变化，由于血液所需的嗜中性粒细胞隔离的体积只允许有一个样品甚至常常需要汇集血液从多个动物为一个单一的测定，例如，涉及的研究米在多个时间点ultiple条件和治疗方法可以使用​​目前的趋化实验可能需要上千小鼠。这限制了基础生物学研究是可以做到的，了解的损伤，感染的情况下免疫功能的复杂动态或烧伤经常研究在小鼠模型5。 以满足对嗜中性粒细胞功能测定法是快速，健壮，同时要求最小的血液量，我们已经开发了直接从一个小液滴的全血测定中性粒细胞趋化性的微流体装置。它是已知的许多因素中的全血，血清，包括6和血小板7，影响嗜中性粒细胞的功能。因此，它是有益的，该全血的微流体测定最大限度地减少样品处理测量的变化趋当与体外测定8维持体内微环境中的中性粒细胞。这种方法降低时间从采血到使用传统技术时中性粒细胞迁移实验，以短短几分钟内（ 表1）。全血的微流体平台产生用于实验的长度的稳定的线性趋化梯度，没有移动部件，并且不需要外部压力源（ 例如注射器泵）。在全血中的微流体设备的设计中的关键特征是红细胞（RBC）的过滤梳机械地过滤进入设备的迁移通道的RBCs的掺入。这个过滤梳的右转弯防止需要排阻过滤，这将可能由红细胞堵塞，因此到达在WB的积极迁移阻断中性粒细胞趋化梯度。全血的微流体装置的一个12或24孔板的掺入促进人或鼠中性粒细胞趋化性的Si多种介质的筛分multaneously。

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			<td height="42" style="height:42px;width:211px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:179px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Device Fabrication</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">SU8&#160;</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:179px;">Y131273</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:191px;">Ellsworth Adhesives</td>
			<td style="width:179px;">Sylgard 184 1.1 lb. Kit</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Standard glass slides</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">125495</td>
			<td style="width:191px;">1 X 3 inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Glass-bottom plate</td>
			<td style="width:191px;">MatTek</td>
			<td style="width:179px;">P12G-1.5-14-F</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 5.0mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15081</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 1.5 mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15072</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Microfluidic Assay Preparation and Analysis</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Gel-loading pipet tip</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">02-707-139</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Syringe</td>
			<td style="width:191px;">Fisher Scientfic</td>
			<td style="width:179px;">309602</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Blunt tip needle, 30g &#189; in.</td>
			<td style="width:191px;">Brico Medical Supply</td>
			<td style="width:179px;">BN3005</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Vacutainer, Heparin</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">HBSS&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Human serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A5843-5G</td>
			<td style="width:191px;">0.2% final concentration in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fibronectin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F0895-1MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">fMLP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F3506-10MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">SurgiLance safety lancet, 2.2mm depth, 22 gauge</td>
			<td style="width:191px;"></td>
			<td style="width:179px;">SLN240</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Hoescht stain</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:179px;">H3570</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Positive Control&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HetaSep</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">7906</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">EasySep Human Neutrophil Enrichment Kit</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">19257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Plasma Asher</td>
			<td style="width:191px;">March Instruments</td>
			<td style="width:179px;">P-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Lindberg/Blue M Oven</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:179px;">13-258-30C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Stainless Steel Precision Tweezers</td>
			<td style="width:191px;">Techni Tool</td>
			<td style="width:179px;">758TW458</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Bel-Art Scienceware Chemical-Resistant Vacuum Desiccator</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">08-594-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Dataplate Digital Hot Plate</td>
			<td style="width:191px;">Alpha Multiservices</td>
			<td style="width:179px;">PMC 720&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Nikon TiE inverted microscope</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MEA53100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">CFI Plan Fluor DL 10x na 15.2wd Objective</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MRH20101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Lumen 200 with 2 Meter Light Guide for Nikon</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">500-L200NI2</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:211px;">DAPI/Hoechst/AMCA Narrow Band 32mm Exciters - 25mm Emitters</td>
			<td style="width:191px;">Chroma - Micro Video Instruments Inc.</td>
			<td style="width:179px;">31013v2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Retiga R 2000 cooled CCD Camera 1600x1200 pixels</td>
			<td style="width:191px;">Qimaging - Micro Video Instruments Inc.</td>
			<td>RET-2000R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood

中性粒细胞在抵御感染和它们的数量在血液中发挥至关重要的作用，经常测量在诊所。在血液中较高的中性粒细胞计数通常持续感染的指标，而低中性粒细胞计数是一个警告标志的感染风险较高。为了实现其职能，中性粒细胞也必须能够有效地从那里度过大部分生命的血液移动，进入组织，在感染发生。因此，在嗜中性粒细胞的迁移能力的任何缺陷可以提高风险的感染，即使在中性粒细胞是存在于血液中的相应的数字。但是，衡量在诊所中性粒细胞迁移能力是一项艰巨的任务，这是费时，需要大量的血液，和专家知识。为了解决这些限制，我们设计了一个强大的微流体检测中性粒细胞的迁移，这需要未处理血单个液滴，circum气孔的必要性嗜中性粒细胞分离，并且容易量化的简单的显微镜。在该测定中，嗜中性粒细胞直接从血液滴，迁移通过小通道朝趋化因子的来源。为了防止通过同一信道的红血细胞的颗粒流，我们实现了机械过滤器具有直角转弯，其选择性地阻挡红细胞的前进。我们通过从手指刺和静脉血采集的血液滴比较中性粒细胞迁移的验证实验。我们还比较了这些全血（WB）的来源与中性粒细胞纯化样品中性粒细胞迁移，发现三个来源之间保持一致的速度和方向。此微流体平台将使人类中性粒细胞迁移的研究在临床和研究环境，以帮助增进我们的健康和疾病中性粒细胞功能的理解。

全血（WB）中性粒细胞的趋化性试验，通过测量中性粒细胞朝着渐变的fMLP（ 动画S1）的积累验证。结果证实，红细胞通过的过滤梳困而嗜中性粒细胞（蓝色）能够积极地迁移出的全血（ 图3A和电影S1）。由全血的微流体装置所形成的稳定的线性趋化梯度（绿色）用FITC-标记的葡聚糖（ 图3A中的插图）的确定和测量的荧光水平随着时间的推移。在这?...

Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

这个协议的细节设计，从全血具有强大的可重复性一滴测量人体中性粒细胞的趋化作用的试验。这种方法绕过了必要的中性粒细胞的分离和需要测定的准备时间仅几分钟。微流控芯片使中性粒细胞的趋化作用的重复测量随着时间的推移在婴儿或小型哺乳动物，其中样品体积是有限的。

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Research

JoVE Journal

Bioengineering

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...