Name: methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps
Uploaded: 2017-03-29T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps at JoVE.com

由Akademie的献给Tiergesundheit（AFT）和博士课程，奖学金的奖学金"动物和人畜共患病，"兽医，德国汉诺威大学，提供给阿恩·纳曼的这项工作得到了支持。

外周血中此协议的集合是经当地人类研究伦理委员会。所有的人类受试者提供书面知情同意书。 1.通过密度梯度离心分离人血液来源的中性粒细胞<ol><li> 人血源性中性粒细胞的分离 <ol><li>层〜20毫升的血液20上毫升附近的火焰和没有混合泛影钠/葡聚糖溶液。 </li><li>离心机在470×g离心30分钟，不制动。 </li><li>除去单核细胞和血浆淡黄色层。传送多形核细胞（PMN）相（第2相具有累积的细胞; 图1和图 2）到一个新的50毫升管中并填充它高达50毫升用1X磷酸缓冲盐水（PBS）。 </li><li>离心机在470×g下与制动器10分钟。 </li><li>去除上清，重悬沉淀在5毫升的无菌水中FO个R 5以裂解红细胞。 </li><li>立即填充至50mL用1×PBS和离心机在470×g下10分钟。 </li><li>除去上清液。沉淀应该是白色。如果它仍然是红色，重复步骤1.1.5和1.1.6。 </li><li>在重悬的Roswell Park纪念研究所（RPMI）中的1000微升沉淀。使用台盼蓝染色用血细胞计数器在光学显微镜下计数细胞数目。 </li><li>以2×10 6 / mL的浓度制备在RPMI细胞悬浮液。约2.5×10 7嗜中性粒细胞可以从20毫升的血液中收获。 </li></ol></li><li> 嗜中性粒细胞脂质分析的药物治疗 <ol><li>使用5×10 7细胞从步骤1.1.9。调整在300μL的在1.5mL反应管的总体积中在10mMMβCD或25nM的PMA的存在或不存在纯Hank&#39;s平衡盐溶液（HBSS）中的细胞数。 </li><li>孵育的样品反应管在37℃下2小时和5％CO 2。 </li><li>离心机在470×g下与制动器10分钟。 </li><li>除去上清，悬浮于HBSS中的300μL细胞沉淀。 </li><li>离心机在470×g下与制动器10分钟。 </li><li>通过进出到1mL注射器10倍抽吸样品，用26-G套管之均匀，将样品储存在-20℃:重悬在1mL氯仿/甲醇的细胞沉淀（11）。 </li></ol></li><li> 嗜中性粒细胞的药物治疗，免疫荧光定量NET <ol><li>制备聚-L-赖氨酸包被的48孔板用玻璃盖玻片。 <ol><li>放置一个8毫米的玻璃盖玻片在各孔中，添加无菌0.01％聚-L-赖氨酸的55μL，并孵育〜在室温（RT）20分钟。 </li></ol></li><li>用1x PBS的200微升洗两次。存储与每孔200μL的1×PBS中的板在冰箱中，直到需要。 </li><li>汽车efully从步骤1.1.9每孔在每个盖玻片的中心加入100微升细胞悬浮液（2×10 5 /100μL）的。 </li><li>添加每任一最终的10mMMβCD或最终25nM的PMA的孔100μL。 </li><li>离心机在370×g下与制动器5分钟。 </li><li>孵育在37℃下2小时和5％CO 2。 </li><li>离心机在370×g下与制动器5分钟。 </li><li>固定细胞用PFA 155μL的4％，在塑料薄膜石蜡包装它们，并把它们在4℃下或在室温下10分钟过夜储存。 注:有关由MβCD诱导NET形成的数据，参见Neumann 等。 15。 </li></ol></li></ol> 2.脂质分离和人血源性中性粒细胞的分析<ol><li> 隔离基于Bligh和Dyer的22和Brogden 等人外周血来源粒细胞中的脂质。 23 <ol><li&gt;从步骤1.2.6以嗜中性粒细胞，将它们放在冰上。 </li><li>在冰上，移液管将嗜中性粒细胞（存在于甲醇和氯仿溶液）至15mL螺旋盖玻璃管用聚四氟乙烯（PTFE）密封件和通过摇动1分钟均质化它们。用玻璃管，以防止脂质的结合到塑料表面。使用PTFE上限，以防止从橡胶/塑料污染。 </li><li>加甲醇的2mL的随后1分钟后加入1毫升氯仿中。 1分钟再摇晃。 </li><li>旋转在RT玻璃管和50rpm下30分钟。 </li><li>通过离心在7℃下的溶液中，1952×g离心10分钟，沉淀蛋白级分。 </li><li>小心倒出上清液到新的15个姆·格拉斯螺旋盖管中，留下含有蛋白质的沉淀物的后面。在-20℃下以供将来量化存储沉淀。 </li><li>加入1毫升氯仿中，等待1分钟，加入1毫升的双蒸水，和与样品30秒反转玻璃螺丝帽管中。 </li&gt; <li>离心机在7℃和1952×g离心10分钟并弃去上层相，向下，但不包括该浑浊层。 </li><li>如果需要，执行由重复步骤2.1.8任选进一步的纯化步骤。 </li><li>在真空中干燥浓缩样品在60℃，并将其在-20存储℃直至需要。 </li></ol></li><li> 高效薄层层析（HPTLC）到半量化几个脂质，包括胆固醇，磷脂，鞘磷脂和的量 <ol><li>准备以下四个运行的解决方案和染色溶液。 <ol><li>溶液1:将乙酸乙酯（26.6％），1-丙醇（26.6％），氯仿（26.6％），甲醇（26.6％），和氯化钾（9.6％）。通过0.25克氯化钾溶解在100mL HPLC级水制备的KCl。 </li><li>溶液2:将正己烷（73％），乙醚（23％）和柠檬酸（2％）。 </li><li>解决方案3:100％正己烷。 </li><li>准备用染色液与7.5克硫酸铜混合蒸馏水（90mL）中，然后添加10mL的磷酸。 </li></ol></li><li>填充在单独的玻璃腔室的各溶液为5毫米。添加任何一种过滤纸，以提高运行速度。在每个腔室。 </li><li>直到运行溶液到达板的顶部预孵育在所述第一运行溶液20×10cm的HPTLC硅胶60玻璃板。然后干燥它在110℃10分钟。 注意:这些板可以被存储在铝箔将来使用，并且在110℃下在使用前干燥10分钟。 </li><li>溶于氯仿/甲醇的200μL从步骤2.1.10中获得的脂质颗粒（1:1）溶液中并在37℃孵育15分钟以溶解。 </li><li>用尺子和软铅笔标记样品所需数量的装载点，再加上至少一个标准。标记的行驶距离在约4cm和6厘米为第一和第二行驶溶液，分别。 </li><li>加载样品，洗10μL注射器3倍于氯仿/甲醇（1:1）加载的每个新样品之前。负载10μL各样品逐滴的，试图专心小的面积尽可能的样品。样品应至少一式两份加载。 </li><li>放置板垂直地进入第一腔室与运行溶液1（步骤2.2.1.1）。确保板平行于该玻璃室的壁以获得均匀的迁移速度。 </li><li>一旦溶剂线已达到第一标记，取出板，甩干，并将其放置在所述第二溶液中。重复第二和第三行驶的解决方案类似的步骤;离开板在溶液中直到溶剂前沿到达板的顶部，然后进行1分钟取出，干燥它在室温。 </li><li>置于硫酸铜溶液板7秒。取出板，彻底干燥，并烘烤的烘箱中在170℃下7分钟。等待板冷却。 </li><li> USI纳克薄层色谱法和凝胶分析的软件，以及一个图象处理软件，扫描和通过Brogden 等人先前所描述的分析。 23。 </li></ol></li><li> 高效液相色谱法（HPLC）定量胆固醇的量 <ol><li>附加一个100×4.6mm柱，以5微米/4.6毫米保护柱和其加热到32℃。使用甲醇作为流动相在65巴的1毫升/分钟的流速和在202纳米的UV检测器测量来定量各样品中胆固醇的量。 </li><li>洗HPLC机器之前彻底分析样品，执行以下步骤:清洗泵，洗涤针，漂洗罐，吹扫注射器，和洗涤泵净化。用清水洗净所有。最后，以5mL / min的流速为5分钟执行与甲醇的系统吹扫。 </li><li>建立了胆固醇的标准曲线，制备至少4个浓度范围为0.05毫克/毫升至2mg /胆固醇在氯仿/甲醇中的溶液（1:1）24。 </li><li>重悬在500微升的氯仿/甲醇中，从步骤2.1.10中获得的样品（1:1）与螺旋盖的红色PTFE /白色硅胶盖的琥珀色1.5毫升玻璃瓶中。 </li><li>量化使用在步骤2.3.3制备的标准中的胆固醇浓度。 注:填写采样协议，从至少一个标准和一个阴性对照，接着是样本。 </li><li>表示结果的曲线下面积和使用任何统计软件合适的样品之间进行比较。 注:测定结果，也可以根据标准曲线定量并且可以被表示为每毫升中的总胆固醇量，g或细胞的数目。标准曲线方程应当通过使用在步骤2.3.3中获得的值来计算。 </li></ol></li></ol>教师的3.可视化和量化<ol><li> 可见教师的ualization 注:网的可视化是基于Neumann 等以前发表的作品。 15。 <ol><li>从步骤1.3.8 3次用1×PBS的200μL洗涤固定的样品。 </li><li>块和用100μL2％BSA，0.2％的Triton X-100透化在PBS中，每孔在RT下45分钟。 </li><li>加入第一抗体的100μL:小鼠单克隆抗DNA组蛋白1抗体（稀释股票与2.2毫克/毫升1:5000在含有2％BSA和0.2％PBS的Triton X-100）或针对髓过氧化物酶的多克隆抗体（MPO;兔抗MPO;稀释1:在含有2％BSA和0.2％的Triton X-100）的PBS 300。在4℃下孵育过夜。 </li><li>用1×PBS的200μL洗涤3次。 </li><li>添加100μL的次级抗体（荧光标记的山羊抗 - 小鼠; 1:1000在BSA-PBS-的Triton X-100和山羊抗 - 兔; 1:1000 BSA-PBS-的Triton X-100）在1个小时RT在黑暗中。 </li><li>用200＆＃洗3次181，L 1×PBS中。 </li><li>使用镊子除去盖玻片并面朝下与含在载玻片上DAPI封固剂的3μL。 </li><li>检查使用装备有PL HCX APO 40X 0.75-1.25油浸物镜15共焦倒基础荧光显微镜的样品。 </li></ol></li><li> 净释放核的定量 <ol><li>打开图像处理软件（ 例如，ImageJ的）和拖放感兴趣的图片放到工具栏。 </li><li>点击"插件"，"分析"和"细胞计数"。 </li><li>按在计数器窗口中的"初始化"按钮并选择计数器类型（ 例如，点击7红色NET-释放细胞和8以黄色非释放细胞，如显示于图6B-I和II）。 注:NET阳性细胞的标准:积极染绿核+密度较小胞核S（分叶的损失）或核的圆形形状+核的尺寸增大，或一个不同的胞外偏离拍的发生的损失。 </li><li>点击每一个细胞附着于上述标准，写所计数的细胞数成分析软件的一个数据表。 </li><li>计算使用NET-释放和非释放细胞的计数的细胞数NET-释放细胞的百分比。 </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can J Biochem Physiol. 37, 911-917 (1959).</li><li>Brogden, G., Propsting, M., Adamek, M., Naim, H. Y., Steinhagen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+analysis+of+membrane+lipids+and+lipid+rafts+in+common+carp+(Cyprinus+carpio+L).">Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L).</a> Comp Biochem Physiol B Biochem Mol Biol. 169, 9-15 (2014).</li><li>Saldanha, T., Sawaya, A. C., Eberlin, M. 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B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cast+NETs:+Neutrophil+extracellular+traps+are+released+from+fish+neutrophils.">Fish cast NETs: Neutrophil extracellular traps are released from fish neutrophils.</a> Dev Comp Immunol. 31 (8), 805-816 (2007).</li><li>Ng, T. H., Chang, S. H., Wu, M. H., Wang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrimp+hemocytes+release+extracellular+traps+that+kill+bacteria.">Shrimp hemocytes release extracellular traps that kill bacteria.</a> Dev Comp Immunol. 41 (4), 644-651 (2013).</li><li>Hawes, M. C., 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=Extracellular+DNA:+the+tip+of+root+defenses?.">Extracellular DNA: the tip of root defenses?.</a> Plant Sci. 180 (6), 741-745 (2011).</li><li>Brinkmann, 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=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin?.">Neutrophil extracellular traps: is immunity the second function of chromatin?.</a> J Cell Biol. 198 (5), 773-783 (2012).</li><li>Xu, 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=Lipid+raft-associated+&#946;-adducin+is+required+for+PSGL-1-mediated+neutrophil+rolling+on+P-selectin.">Lipid raft-associated &#946;-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.</a> J Leukoc Biol. 97 (2), 297-306 (2015).</li><li>Ermert, D., 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=Mouse+neutrophil+extracellular+traps+in+microbial+infections.">Mouse neutrophil extracellular traps in microbial infections.</a> J Innate Immun. 1 (3), 181-193 (2009).</li><li>Metzler, K. 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作者什么都没有透露。

这里描述的方法可用于分析特定脂质，如胆固醇，通过HPTLC或HPLC，并调查药理脂质改变对教师的形成的影响（参见Neumann 等15）。 HPTLC是分析大量样品的宽范围的脂质的相对成本有效且简单的方法。该方法已在许多研究领域，包括抗生素量化25，在溶酶体贮积症26脂质贮积，和胆固醇和cholesterylglucoside水平的上皮细胞<...

<html>脂质已经被证明在细胞稳态，细胞死亡，宿主-病原体相互作用，和细胞因子释放1〜发挥重要作用。随着时间的推移，利息和知识脂质在宿主 - 病原体相互作用或炎症的影响有所增加，一些出版物确认某些脂质的核心作用，尤其是类固醇胆固醇在细胞反应。他汀类药物，其通过阻断3-羟基-3-甲基 - 辅酶-A还原酶用作胆固醇生物合成的抑制剂的药理学治疗（HMG辅酶A还原酶），可以通过降低白细胞介素的血清水平充当抗炎剂6和C反应蛋白2。胆固醇和鞘糖脂富集结构可以通过几种病原体，如细菌和病毒一起使用，作为一个网关到主机3，4，5，类= "外部参照"&gt; 6。鞘脂（ 例如，鞘磷脂）已经显示出由病原体被用来促进其致病性7。在巨噬细胞，分枝杆菌使用富含胆固醇的用于输入单元域;胆固醇的耗尽抑制分枝杆菌的摄取8。此外， 土拉弗朗西斯菌，负责兔热病人畜共患剂（也被称为兔热病）9，巨噬细胞的感染导致当胆固醇从膜10耗尽该被废除的感染。类似地，通过富含脂质的结构的宿主细胞大肠杆菌的侵袭被证明是胆固醇依赖性4。此外，上皮细胞的鼠伤寒沙门氏菌感染的实验表明，胆固醇是病原体进入细胞11是必不可少的。胆固醇消耗明显抑制ð沙门氏菌11的摄取。此外，最近的一项研究Gilk 等 。表明，胆固醇起着贝氏burnetti 12摄取了重要的作用。此外，TUONG 等 。发现，25-羟基胆固醇起着由脂多糖（LPS）在吞噬作用至关重要的作用刺激的巨噬细胞13。当巨噬细胞药理学上处理以消耗胆固醇14吞噬功能降低。因此，胆固醇和其他脂质似乎起到感染和炎症的重要作用，因为它们消耗可以从多种病原体10，11，12减少入侵的风险。 最近，我们能够证明脂质的改变，尤其是从细胞胆固醇消耗，诱导中性粒细胞产胞外的形成ř陷阱（母语）在人血液来源的嗜中性粒细胞15。由于网在2004年发现的，它们已被证明在细菌截留中发挥重要作用，从而阻碍在16感染，17的蔓延。母语由具有组蛋白，蛋白酶，和抗微生物肽16相关联的DNA主链的。的母语的嗜中性粒细胞的释放可通过入侵病原体18,19和化学物质如佛波醇肉豆蔻酸乙酸酯（PMA）或他汀类药物16，20进行诱导。然而，详细的细胞机制，尤其是在这个过程中脂类的作用，仍然是不完全清楚。脂质的分析可能会导致更好地了解参与多种细胞过程和相互作用，如教师的释放机制。 CholesteROL和鞘磷脂是细胞膜脂质微，在那里他们增加稳定性，并促进参与蛋白质运输和信号事件21蛋白的聚集的重要组成部分。调查某些脂质，两亲性的药理学试剂，如环状寡糖甲基β环糊精（MβCD）的机械作用，可用于改变细胞的脂质组成，并减少在体外 15胆固醇。这里，我们提出的方法使用HPTLC分析嗜中性粒细胞的脂质组合物响应于MβCD。采用高效液相色谱法确认胆固醇在嗜中性白细胞群体的水平。此外，我们描述可视化响应于MβCD教师的在人血液来源的嗜中性粒细胞免疫荧光显微镜的形成的方法。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Neutrophil isolation, NET staining and quantification&lt;/strong&gt;</td></tr><tr><td>Alexa Flour 633 goat anti-rabbit IgG</td><td>Invitrogen</td><td>A-21070</td><td></td></tr><tr><td>Anti-MPO&amp;alpha; antibody</td><td>Dako</td><td>A0398</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>3912-100G</td><td></td></tr><tr><td>Marienfeld-Neubauer improved counting chamber</td><td>Celeromics</td><td>MF-0640010</td><td></td></tr><tr><td>Confocal microscope TCS SP5 AOBS with tandem scanner</td><td>Leica</td><td>DMI6000CS</td><td></td></tr><tr><td>Dulbecco&amp;acute;s PBS 10x</td><td>Sigma-Aldrich</td><td>P5493-1L</td><td>Dilute 1:10 in water for 1x working solution</td></tr><tr><td>Dy Light 488 conjugated highly cross-absorbed</td><td>Thermo Fisher Scientific</td><td>35503</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td>2010</td><td></td></tr><tr><td>DNA/Histone 1 antibody</td><td>Millipore</td><td>MAB3864</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>1.8</td><td>http://imagej.nih.gov/ij/</td></tr><tr><td>Light microscope</td><td>VWR</td><td>630-1554</td><td></td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Sigma-Aldrich</td><td>C4555-1G</td><td></td></tr><tr><td>PFA</td><td>Carl Roth</td><td>0335.3</td><td>dissolve in water, heat up to 65 &amp;deg;C and add 1 N NaOH to clear solution</td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139-1MG</td><td>Stock 16 &amp;micro;M, dissolved in 1x PBS</td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Polymorphprep</td><td>AXIS-SHIELD</td><td>AN1114683</td><td></td></tr><tr><td>ProLong Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P7481</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Reagent</td><td>Invitrogen</td><td>P7581</td><td></td></tr><tr><td>RPMI1640</td><td>PAA</td><td>E 15-848</td><td></td></tr><tr><td>HBSS with CaCl and Mg</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-50ml</td><td></td></tr><tr><td>Trypanblue</td><td>Invitrogen</td><td>15250-061</td><td>0.4% solution</td></tr><tr><td>Water</td><td>Carl Roth</td><td>3255.1</td><td>endotoxin-free</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Lipid isolation and analysis&lt;/strong&gt;</td></tr><tr><td>1-propanol</td><td>Sigma-Aldrich</td><td>33538</td><td></td></tr><tr><td>10 &amp;micro;L syringe</td><td>Hamilton</td><td>701 NR 10 &amp;micro;l</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma-Aldrich</td><td>346136</td><td></td></tr><tr><td>Ethyl acetate</td><td>Carl Roth</td><td>7336.2</td><td></td></tr><tr><td>Canullla 26 G</td><td>Braun</td><td>4657683</td><td></td></tr><tr><td>Copper(II)sulphatepentahydrate</td><td>Merck</td><td>1027805000</td><td></td></tr><tr><td>Chloroform</td><td>Carl Roth</td><td>7331.1</td><td></td></tr><tr><td>CP ATLAS software</td><td>Lazarsoftware</td><td>2.0</td><td></td></tr><tr><td>Chromolith HighResolution RP-18 endcapped 100-4.6 mm column</td><td>Merck</td><td>152022</td><td></td></tr><tr><td>High Performance Liquid Chromatograph Chromaster</td><td>Hitachi</td><td>HITA 892-0080-30Y</td><td>Paramaters are dependent on individual HPLC machine</td></tr><tr><td>HPLC UV Detector</td><td>Hitachi</td><td>5410</td><td></td></tr><tr><td>HPLC Column Oven</td><td>Hitachi</td><td>5310</td><td></td></tr><tr><td>HPLC Auto Sampler</td><td>Hitachi</td><td>5260</td><td></td></tr><tr><td>HPLC Pump</td><td>Hitachi</td><td>5160</td><td></td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>7342.1</td><td></td></tr><tr><td>n-Hexane</td><td>Carl Roth</td><td>7339.1</td><td></td></tr><tr><td>Phosphoric acid</td><td>Sigma-Aldrich</td><td>30417</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>49,361,000</td><td></td></tr><tr><td>Potters</td><td>LAT Garbsen</td><td>5 ml</td><td></td></tr><tr><td>SDS</td><td>Carl Roth</td><td>CN30.3</td><td></td></tr><tr><td>HPTLC silica gel 60</td><td>Merck</td><td>105553</td><td></td></tr><tr><td>Vacufuge plus basic device</td><td>Eppendorf</td><td>22820001</td><td></td></tr><tr><td>Corning Costar cell culture 48-well plate, flat bottom</td><td>Sigma</td><td>CLS3548</td><td></td></tr><tr><td>Coverslip</td><td>Thermo Fisher Scientific</td><td>1198882</td><td></td></tr><tr><td>Glass slide</td><td>Carl Roth</td><td>1879</td><td></td></tr><tr><td>BD Tuberculin Syringe Only 1 mL</td><td>BD Bioscience</td><td>309659</td><td></td></tr></tbody></table>

methods to study lipid alterations in neutrophils and the subsequent formation of neutrophil extracellular traps

通过高效薄层层析（HPTLC）进行脂质分析是分析各种脂质的相对简单的，成本有效的方法。脂质（ 例如，在宿主-病原体相互作用或主机条目）的功能已被报道在细胞过程中起关键作用。在这里，我们示出了方法来确定脂质组合物，具有相比于高效液相色谱法（HPLC）的聚焦初级血液衍生的中性粒细胞的胆固醇水平，通过HPTLC。的目的是调查脂质/胆固醇改变的嗜中性粒细胞的胞外陷阱（母语）的形成中的作用。 NET版本被称为宿主防御机制，以防止病原体的宿主内蔓延。因此，血液来源的人中性粒细胞用甲基β环糊精（MβCD）处理以诱导脂质改变的细胞。使用HPTLC和HPLC，我们已经表明，MβCD处理的细胞导致脂质与小区中的胆固醇含量降低显著相关联的改造。与此同时，MβCD治疗嗜中性粒细胞的导致母语的形成，如通过免疫荧光显微术。总之，在这里我们提出一个具体的方法来研究在嗜中性粒细胞脂质改变和渔网的形成。

人血液来源的嗜中性粒细胞通过密度梯度离心（ 图2）中分离。为了研究嗜中性粒细胞上的脂质改变的效果，将细胞用10mMMβCD，其从细胞耗尽胆固醇处理。随后，将脂质从通过Bligh和Dyer的（ 图1，左图）的样品中分离，如通过Brogden 等人描述。 23。将制备的脂质样品加载到使用三溶液协议，它已被优化，以分离和可视...

Watch this Scientific Journal Video about Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps at JoVE.com

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

脂质是已知在细胞功能中起重要作用。在这里，我们描述了一种方法，以确定嗜中性粒细胞的脂质组合物，重点对胆固醇水平，同时使用HPTLC和HPLC纯化，获得更好的理解嗜中性粒细胞的胞外圈闭形成的基本机制的。

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 ...

methods-to-study-lipid-alterations-neutrophils-subsequent-formation

Research

JoVE Journal

Immunology and Infection

9.4K 次观看.  University of Veterinary Medicine Hannover. 脂质是已知在细胞功能中起重要作用。在这里，我们描述了一种方法，以确定嗜中性粒细胞的脂质组合物，重点对胆固醇水平，同时使用HPTLC和HPLC纯化，获得更好的理解嗜中性粒细胞的胞外圈闭形成的基本机制的。 

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

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

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 ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

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 ...

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 ...

使用双染料活细胞分析定量 NET 形成

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

作者聚焦：使用双色活细胞成像定量疾病中的中性粒细胞细胞外陷阱和药物筛选

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

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

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嗜中性粒细胞胞外的陷阱：如何生成和可视化

利用共焦显微镜可视化巨噬细胞胞外陷阱

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

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

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成