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毫升淋巴细胞分离介质（LSM）的。使用18G的针头装在60毫升注射器，仔细一层稀释血液到LSM，创造一个尖锐的LSM-血接口。 注：避免混合层尽可能。 </li><li>离心机在800 XG为30分钟，在21°C不休息。 注：突然断裂能引起的不同层的混合。 </li><li>观察红细胞和嗜中性粒细胞沉淀在底部。吸并丢弃顶2层（血浆和LSM）确保这些层包含淋巴细胞和单核细胞之间摆脱了接口，只留下底部的红层。 </li><li>向每个管中，加入20毫升磷酸盐缓冲液盐水（PBS）和20毫升6％葡聚糖溶液。轻轻颠倒每个管混合与红细胞和嗜中性粒细胞的层，并允许坐在RT下30分钟。 注意：这将允许红血细胞（红细胞），以沉淀在底部。 </li><li> 30分钟后，转移的嗜中性粒细胞丰富上清到新鲜试管，弃去红细胞粒料。离心上清在450×g离心在4℃下5分钟。离心后，弃去上清液。颗粒将包含多为中性粒细胞和红细胞数。 </li><li>通过加入0.5ml裂解缓冲液至4.5毫升的无菌水的制备裂解液。裂解剩余RBC伴t他准备5毫升裂解液，将全部再悬浮颗粒进入一管。允许裂解在室温在黑暗中进行10分钟。 </li><li>离心机在450×g离心在4℃下5分钟。弃上清，用5毫升PBS中再在450×g离心在4℃下洗涤沉淀不含Ca和Mg和离心机摆脱任何剩余裂解溶液。在这点上得到的粒料中含有的嗜中性粒细胞。悬浮颗粒在30毫升冷的3％RPMI和雪藏。 注：3％的RPMI由补充RPMI培养基与由3％胎牛血清（FBS）的</li><li>验证使用亚甲蓝染色样品的纯度。 &gt; 95％的细胞应粒具有多叶核。用台盼蓝染色，以验证&gt;细胞95％的生存能力和计算最终的中性粒细胞的产量。稀释在冰冷的3％的RPMI嗜中性粒细胞，以获得5×10 6中性粒细胞/ ml的终浓度。 </li></ol> 3.英语教师代<ol><li>刺激中性粒细胞与500 NPMA的M个（每30毫升的中性粒细胞的溶液），并孵育在150×25毫米平组织培养皿用20毫米的网格4小时，在37℃，5％CO 2。这将允许NETosis。 </li><li> 4小时的刺激后，轻轻吸并丢弃媒体，留下网和嗜中性粒细胞粘附在底部的层。不要破坏这层。 </li><li>使用总共15毫升冷的PBS无钙和镁每皿，通过以抬离所有附着的物质从底部吹打在培养皿的底部15毫升的PBS冲洗每个培养皿的底部。 </li><li>收集来自在450×g离心在15ml锥形管中，并离心洗涤每个培养皿（步骤3.3）进行10分钟，在4℃下得到的溶液中。嗜中性粒细胞和任何剩余的细胞将沉淀在底部，留下无细胞的NET-富上清液。 </li><li>除法上清液进1.5毫升微离心管中，自旋10分钟，在18000×g离心在4℃下。这将允许所有的DNA沉淀。 注：离心较大TUBES是更容易和耗时更少如果这样高的速度是可以实现在常规离心机，否则将需要划分在较小的管的上清液，以便使用高速微离心机。 </li><li>弃上清，重悬所得一起在冰冷PBS到对应于每100微升的PBS的2×10 7嗜中性粒细胞的浓度的所有颗粒。这将产生的无细胞NET股票，可以用于后续的实验。 </li><li>测量DNA浓度用分光光度法或候补DNA的定量工具得到的样品中。样品中的适当浓度应介于140 - 180纳克/微升。 </li></ol> 4. NET-癌细胞静态粘附实验<ol><li>在黑暗中添加100微升每孔的以前取得的净股票在96孔平底板，并允许以涂覆孔O / N在4℃。 </li><li>后12至20小时，确认形成细胞均匀单层在孔的显微镜下的底部自由母语（ 图1）。在这一点上，轻轻地吸出所有非粘附材料从孔中，确保不破坏网的单层的底部。 </li><li>加入100微升的1％牛血清白蛋白（BSA）的封闭溶液以每孔并离开在RT 1小时。 </li><li>从T-75烧瓶中使用2-毫升0.25％胰蛋白酶溶液中分离的A549癌细胞。一旦分离，加入10 mL的A549媒体来胰蛋白酶细胞和离心机在450×g离心在4℃下5分钟。弃上清，重悬细胞培养基，以获得2×10 4个 ，每100微升培养基的癌细胞的浓度。 注：A549细胞分别生长。简言之，将细胞培养，并保持在含有10％FBS和1％青霉素链霉素，37℃，5％CO 2的DMEM F12培养基。一旦70％ - 80％的细胞汇合达到，他们使用0​​.25％胰蛋白酶-EDTA进行分离，并重新悬浮在相同的媒体如上所述。 </li><li>用CFSE通过加入1微升每ml培养基CFSE和离开染色在室温10分钟染色癌细胞。染色，离心下来，在450×g离心在4℃下5分钟后，然后弃上清，重悬细胞的培养基的初始体积，以获得2×10 4个 ，每100微升培养基的癌细胞的浓度。 </li><li> 1小时后的教师阻断，轻轻吸封闭溶液，并添加2×10 4个肿瘤细胞中的媒体，这相当于100微升，每孔过网单层，并允许在37℃，5％CO 2的粘附90分钟的。轻轻吸出细胞，并添加100微升PBS中，以每个孔洗任何非贴壁细胞。 </li><li>在一些井中，洗涤前DNAse1的添加1000U每孔10分钟以降解母语。这将作为阴性对照。在其他井，10分钟后，将作为车辆控制（VC）的添加100微升每孔的无菌水。 注：3元conditio复制n为通常执行以增加样本量。 </li><li>吸并丢弃在井只剩下网和贴壁肿瘤细胞在底部的所有解决方案。加入100微升每孔的4％甲醛溶液固定粘附癌细胞蚊帐和荧光显微镜下读取测定（ 图1）。打印和分析结果（ 图3）。 </li></ol>

<ol>
	<li>Saitoh, 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=Neutrophil+extracellular+traps+mediate+a+host+defense+response+to+human+immunodeficiency+virus-1.">Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1.</a> Cell Host Microbe. 12 (1), 109-116 (2012).</li><li>Bruns, 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=Production+of+extracellular+traps+against+Aspergillus+fumigatus+in+vitro+and+in+infected+lung+tissue+is+dependent+on+invading+neutrophils+and+influenced+by+hydrophobin+RodA.">Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.</a> PLoS Pathog. 6 (4), e1000873 (2010).</li><li>Brinkmann, V., 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+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Papayannopoulos, 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=NETs:+a+new+strategy+for+using+old+weapons.">NETs: a new strategy for using old weapons.</a> Trends Immunol. 30 (11), 513-521 (2009).</li><li>McDonald, B., Urrutia, R., Yipp, B. G., Jenne, C. N., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+neutrophil+extracellular+traps+capture+bacteria+from+the+bloodstream+during+sepsis.">Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.</a> Cell Host Microbe. 12 (3), 324-333 (2012).</li><li>Pilsczek, F. H., 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=A+novel+mechanism+of+rapid+nuclear+neutrophil+extracellular+trap+formation+in+response+to+Staphylococcus+aureus.">A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.</a> J Immunol. 185 (12), 7413-7425 (2010).</li><li>Villanueva, E., 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=Netting+neutrophils+induce+endothelial+damage,+infiltrate+tissues,+and+expose+immunostimulatory+molecules+in+systemic+lupus+erythematosus.">Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.</a> J Immunol. 187 (1), 538-552 (2011).</li><li>Khandpur, 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=NETs+are+a+source+of+citrullinated+autoantigens+and+stimulate+inflammatory+responses+in+rheumatoid+arthritis.">NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.</a> Sci Transl Med. 5 (178), 178ra140 (2013).</li><li>Demers, 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=Cancers+predispose+neutrophils+to+release+extracellular+DNA+traps+that+contribute+to+cancer-associated+thrombosis.">Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.</a> Proc Natl Acad Sci U S A. 109 (32), 13076-13081 (2012).</li><li>Hahn, S., Giaglis, S., Hoesli, I., Hasler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+NETs+in+reproduction:+from+infertility+to+preeclampsia+and+the+possibility+of+fetal+loss.">Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss.</a> Front Immunol. 3, 362 (2012).</li><li>Cools-Lartigue, 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=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+and+promote+metastasis.">Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis.</a> J Clin Invest. , (2013).</li><li>Cools-Lartigue, J., Spicer, J., Najmeh, S., Ferri, L. <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+cancer+progression.">Neutrophil extracellular traps in cancer progression.</a> Cell Mol Life Sci. , (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., 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=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> J Vis Exp. (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. 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=Sex+hormones,+acting+on+the+TERT+gene,+increase+telomerase+activity+in+human+primary+hematopoietic+cells.">Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.</a> Blood. 114 (11), 2236-2243 (2009).</li><li>Zhong, C., Qu, X., Tan, M., Meng, Y. G., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+regulation+of+bv8+in+human+blood+cells.">Characterization and regulation of bv8 in human blood cells.</a> Clin Cancer Res. 15 (8), 2675-2684 (2009).</li><li>Wu, Y. 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>

作者什么都没有透露

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 ...

simplified-human-neutrophil-extracellular-traps-nets-isolation

Research

JoVE Journal

Immunology and Infection

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

29.5K 次观看.  McGill University. 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。 

视频: Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

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

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

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

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

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

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

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

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

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

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

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 ...

染色和成像以可视化中性粒细胞胞外陷阱

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

作者聚焦：人和小鼠组织中的中性粒细胞胞外陷阱成像 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

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 ...