Name: simplified human neutrophil extracellular traps (nets) isolation and handling
Uploaded: 2015-04-16T13:00:00
Description: Watch this Scientific Journal Video about Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling at JoVE.com

The authors would like to acknowledge Dr. Paul Kubes for his guidance and mentoring that were central in the construction of this work.

注：所有实验均按照当地机构的道德准则进行的。 1.采血<ol><li>通过肘静脉穿刺，取血14管从健康志愿者为青顶（肝素）管和结算冰上反转前每管。在采集血液10 - 实验的15分钟，以保证最佳的中性粒细胞的产量。 </li></ol>从全血中性粒细胞2.隔离<ol><li>洗血每一个绿色的顶管用5毫升的PBS无钙和镁，以稀释血液。在每4×50毫升锥形管中，在室温添加15毫升淋?...

<ol>
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(36), (2010).</li><li>Maqbool, M., Vidyadaran, S., George, E., Ramasamy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+laboratory+procedures+for+isolating+human+peripheral+blood+derived+neutrophils.">Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils.</a> Med J Malaysia. 66 (4), 296-299 (2011).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+lymphocytes,+granulocytes+and+macrophages.">Isolation of lymphocytes, granulocytes and macrophages.</a> Scand J Immunol. Suppl. 5, 9-15 (1976).</li><li>Calado, R. 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J., 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+vivo+leukocyte+labeling+with+intravenous+ferumoxides/protamine+sulfate+complex+and+in+vitro+characterization+for+cellular+magnetic+resonance+imaging.">In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging.</a> Am J Physiol Cell Physiol. 293 (5), C1698-C1708 (2007).</li><li>Saffarzadeh, M., 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+directly+induce+epithelial+and+endothelial+cell+death:+a+predominant+role+of+histones.">Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.</a> PLoS One. 7 (2), e32366 (2012).</li><li>Fuchs, T. A., 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=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176 (2), 231-241 (2007).</li></ol>

在这部影片中表现出的嗜中性粒细胞胞外陷阱隔离协议将在文献中用于中性粒细胞的分离和NET形成不同的技术。它简化了一个相当复杂而多变的过程，并提供了一​​个非常可靠的，可复制的方式来隔离纯化无细胞的教师比其他协议的步骤更少。此外，一应俱全试剂采用增加了协议的简单性和显著降低其成本。教师的朝向一个静态粘附试验中的应用用来表明由该隔离技术所提供的灵活性，因为净?...

中性粒细胞胞外陷阱（英语教师）是新发现的细胞外的DNA链由数百蛋白质和组蛋白的装饰组成的中性粒细胞衍生的元素。它们是由下列具体的刺激在感染和炎症的上下文中的中性粒细胞分泌的，它们最初被认为是对抗感染的嗜中性粒细胞的防御机制的一部分。他们首先证明能促进各种微生物的入侵，包括细菌，病毒和真菌1-3诱捕。然后圈住微生物将其破坏双方直接NET相关蛋白3,4和间接通过吞噬细胞5,6当地招聘领导。然而，似乎教师的角色不局限于宿主防御。最近，它们已显示出在自身免疫性疾病7,8-重要作用，血栓9，妊娠相关疾病10乃至癌的进展11,12。因此，看来母语在大量不同的生理过程中发挥了重要作用。然而，由于这类研究的新特性，仍有许多关于由哪个网络发挥其作用机制阐明。在此，我们提出了一个简单的方法，这些教师在没有嗜中性粒细胞的隔离。 在下面的视频中，我们展示一个简化和简单的技术来隔离人全血的教师。无细胞母语然后可以在多个体外实验包括染色，imuunofluorescence使用，印迹以及许多测定法如粘附，增殖和迁移的。其他协议存在13，19，但它们通常是冗长的，复杂的，昂贵的，并且常常，产量低13。这种简化的协议使用基本试剂和减小的分离嗜中性粒细胞所需的步骤数，因此减少了程序，可以的长度再同时最大限度地提高产量。 以下方法结合不同的技术来粒细胞隔离先前在文献中描述的，以获得一个简单的协议，得到活嗜中性粒细胞的非常纯的样品。如在文献中，静脉血收集在EDTA管中，并在10分钟用来避免中性粒细胞活化14。我们使用淋巴细胞分离介质（LSM）密度梯度离心，从肝素化的全血，改编自由Boyum等人15首先描述的Ficoll密度离心分离法的方法分离粒细胞和红细胞。 LSM是Boyum制剂代替泛影酸钠的甲泛影钠，并已成功地用在许多研究中，分离的中性粒细胞16-18的变形。 下面的微分密度离心，单核细胞和淋巴细胞被丢弃;然后红细胞沉降用6％的葡聚糖溶液14和剩余的红细胞被裂解，以获得纯的中性粒细胞，其与台盼蓝和亚甲基蓝染色证实的群体。 众多剂已被用于诱导NETosis 在体外和体内 ，包括脂多糖（LPS），以及佛波醇肉豆蔻乙酸酯（PMA）和白介素（IL-8）-3,5,6-。在下面的协议中，分离的嗜中性粒细胞刺激与500nM的PMA 4小时，这已被证明是足够的浓度，以允许一致和可靠的净形成而不促进细胞凋亡19,20。 分离后，我们展示了如何从该协议得到的无细胞的教师可以在附着测定中使用。 DNAse1用于消化和如前所述降解母语，因此作为控制2,3,11。其它选项包括使用中性粒细胞弹性蛋白酶抑制剂（NEI）的抑制NET formati上，这是当目标是抑制NET形成而不影响它们的降解11的可接受的替代品。虽然支持间隙具有许多不同的功能，它已被以前曾表明，净沉积可以通过染色质解聚，核脱粒和嗜中性粒细胞死亡12的堵塞来抑制由支持间隙

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:75px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:573px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">311-425-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">305-010-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran hydrochloride (powder)</td>
			<td style="width:75px;">Spectrum</td>
			<td style="width:119px;">DE130</td>
			<td>6% Dextran solution is made by supplementing PBS with Ca and Mg with 6% Dextran powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 Medium with L-glutamine</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">350-000-CL</td>
			<td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BD Pharm Lyse Lysing buffer</td>
			<td style="width:75px;">BD Biosciences</td>
			<td style="width:119px;">555899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td style="width:75px;">Sigma-Alrdrich</td>
			<td style="width:119px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse1</td>
			<td>Roche</td>
			<td style="width:119px;">11284932001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F12 DMEM</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">319-075-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CFSE</td>
			<td style="width:75px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Integrid tissue culture dish with 20mm grid (150 x 25mm)</td>
			<td style="width:75px;">Falcon</td>
			<td style="width:119px;">353025</td>
			<td></td>
		</tr>
	</tbody>
</table>

simplified human neutrophil extracellular traps (nets) isolation and handling

中性粒细胞胞外陷阱（英语教师），最近被确定为中性粒细胞的抗菌医疗设备的一部分。除了它们在对抗感染的作用，最近的研究表明，它们可能涉及许多其他疾病过程，包括癌症的发展。隔离净化网是一个关键因素，让这些功能的研究。 在这段视频中，我们展示无细胞NET隔离从人全血使用现成的试剂的简化方法。孤立母语然后可用于免疫荧光染色，印迹或各种功能测定。这使得他们的生物特性在没有中性粒细胞本身的潜在混杂影响的评估。 密度梯度分离技术是用于分离来自健康供体的全血嗜中性粒细胞。嗜中性粒细胞分离的，然后通过佛波醇12- MYR刺激状态IState 13-乙酸酯（PMA），以诱导NETosis。嗜中性粒细胞活化，然后被丢弃，并且获得无细胞NET库存。 然后，我们说明如何分离母语可以与A549人肺癌细胞的粘附实验中使用。该NET库存被用于涂布的96孔细胞培养板O / N，并确保在孔的底部的适当网单层形成后，CFSE标记的A549细胞加入孔中。贴壁细胞使用的是尼康TE300荧光显微镜量化。在有些井，1000U DNAse1加10分钟计时，以降低母语之前

成功实现用母语静态粘附实验的需要将孔与教师的加成癌细胞之前单层（ 图1）适当的涂层。所有的后续步骤必须谨慎进行不破坏那层。当形成教师的足够层，则有望看到显著癌细胞粘附于母语， 如图2A和2C所示。这种效果是通过加入DNAse1的，这会降低母语如在图2B和2D看出废止。相反地，不应该有显著下降的A549粘附于母语的车辆控制...

Watch this Scientific Journal Video about Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling at JoVE.com

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

在下面的协议中，我们描述了一个非常简单的方法来分离中性粒细胞外使用容易获得的试剂从人全血中的陷阱（母语）。然后，我们说明如何分离的母语可以与癌细胞在体外粘附试验中使用。

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Research

JoVE Journal

Immunology and Infection

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

使用免疫荧光显微镜鉴定帕芬-嵌入式猫动脉血栓中中性粒细胞外陷阱

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

微生物和化学刺激诱导的中性粒细胞胞外陷阱的形态学和组成分析

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

使用改良的夹心 ELISA 定量来自中性粒细胞胞外陷阱的髓过氧化物酶 DNA 和中性粒细胞弹性蛋白酶 DNA 复合物

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues (Video) | JoVE

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...