Name: in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy
Uploaded: 2018-08-24T13:30:00.000+00:00
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Description: Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

相应的作者由莫里斯动物基金会 (D15CA-907) 资助。这项研究得到了加州大学戴维斯分校马健康中心和同伴动物健康中心 (2016-24 F) 的资助。作者想感谢 Geena 的帮助与数字和尼格海谭德阮为她的视频协助。

此处描述的所有方法均经加州大学戴维斯分校机构动物护理和使用委员会批准 (协议编号: 18338)。
1. 收集血液
<ol><li>用注射器吸入21克针, 从头或颈静脉中抽出10毫升的血。</li>
	<li>为了避免过度的剪切应力, 在将血液转入含有肝素的管 (75 USP) 之前, 从注射器中取出针头。轻轻地将管子倒置几次, 以确保抗凝血和血液的充分混合。在将样品放在冰上之前, 检查是否有血块或红细胞聚集物的证据。</li>
</ol>2. 中性粒细胞隔离
<ol><li>稀释抗凝血液与同样数量的冰冷 Dubecco 的磷酸盐缓冲盐水 (DPBS) (pH 值 7.4, 没有价阳离子)。将8毫升稀释的血液转移到50毫升聚丙烯锥管中, 含有25毫升3% 葡聚糖 (从串珠中溶解在0.9% 氯化钠溶液中)。将管子直立在室温下30分钟, 以允许红细胞聚集和沉淀。</li>
	<li>小心5毫升的白细胞血浆 (图 1) 上5毫升的密度梯度介质在14毫升聚丙烯圆底管。小心不要混合2解决方案11。</li>
	<li>离心管在 400 x g与加速度/减速 (A/D) 设置为 0 (没有刹车) 在室温下30分钟。</li>
	<li>使用5毫升血清学吸管通过绕过血浆和外周血单个核细胞 (PBMC) 层来收集粒细胞层 (图 1)。每次使用新的吸管, 尽量减少污染。</li>
	<li>在50毫升聚丙烯锥形管中放置4到6毫升的 polymorphnuclear (中性粒细胞层)。由于少量的红细胞聚集物可以与中性粒细胞层一起吸气, 使用1毫升的血清学吸管轻轻地去除在管底部固定的红细胞聚集物。</li>
	<li>用20毫升的冷纯水溶解剩下的红细胞。在30到六十年代时, 轻轻地将管子多次反转, 以确保足够的株溶藻, 然后再加入相同体积的冰冷1.8% 氯化钠溶液。</li>
	<li>离心机在 112 x g 10 分钟在4°c, A/D 设置为0。</li>
	<li>如果此颗粒呈红色, 重复红细胞溶解步骤。用血清学吸管, 并轻轻地并用重悬100µL DPBS (5 毫米葡萄糖, 0.9 毫米氯化镁2, 1.9 毫米 CaCl2和1% 牛血清白蛋白, pH 7.3) 中的颗粒, 并小心丢弃上清。将所有悬浮细胞结合在冰上。</li>
	<li>使用自动单元分析器执行单元格计数, 并通过 hemocytometer 验证计数。如有必要, 将 PMNs 浓缩到玻璃滑梯上, 使用 cytocentrifuge (1000 rpm, 5 分钟), 并通过计算细胞总数中的嗜中性细胞数来确定中性粒细胞的百分比。</li>
	<li>通过执行 Strober等所概述的台盼蓝排斥试验确定中性粒细胞的生存能力。12成功的隔离应能产生95至100% 的生存能力, 中性粒细胞应大于95%。</li>
	<li>通过将总细胞计数与存活率和中性粒细胞百分比相乘来确定中性粒细胞的浓度。成功的隔离应该会产生从1.5 到 6 x 106/毫升的中性粒细胞浓度。</li>
	<li>稀释中性粒细胞到最后浓度为 1 x 106/毫升与 DPBS 含有5毫米葡萄糖, 0.9 毫米氯化镁2, 1.9 毫米 CaCl2, 和1% 牛血清白蛋白与 pH 调整到7.3。</li>
</ol>3. 活中性粒细胞的荧光显微术
<ol><li>将200至 400 ul 的中性粒细胞 (1 x 106/毫升) 放入聚 l-赖氨酸涂层的12井培养板的每一个井中。允许中性粒细胞通过在37摄氏度处孵化30分钟, 以保持井底。</li>
	<li>将核酸与两种核酸染料之一的1微米标记为一个, 在室温下, 用完整的膜染色的细胞和一个在细胞中沾污的细胞膜 (见材料表), 10 分钟。</li>
	<li>激活100微克/毫升脂多糖 (LPS) 的中性粒细胞 (大肠杆菌O55。B5), 100 nM 佛波 12-酯 13-醋酸盐 (PMA) 为阳性对照, 或等效体积 DPBS 为阴性对照, 180 分钟37摄氏度。</li>
	<li>获取图像使用 GFP (励磁: 470/22 毫微米, 发射: 525/50 毫微米) 和得克萨斯红色渠道 (励磁: 585/29 毫微米, 放射: 628/32 毫微米) 在荧光显微镜在40X 放大率在以下时间点:30, 90, 120 和 180 min。</li>
</ol>4. 利用免疫荧光技术对净成分进行净量化和检测
<ol><li>小心地将18毫米直径的聚 d-赖氨酸涂层盖滑入12井培养板的井中。适当地给水井贴上标签。</li>
	<li>种子100到 200 ul 的孤立中性粒细胞 (1 x 106/毫升) 直接到盖玻片, 并允许中性粒细胞通过孵化的细胞在37°c 30 分钟。</li>
	<li>激活中性粒细胞, 如3.3 所述。用200µΜ氯 amidine (15 分钟37°c) 预处理中性粒细胞抑制网状形成, 如前文所述. 13确保试验中包括适当的车辆控制 (亚砜)。</li>
	<li>小心地取出有细镊子的盖子。在试管架上涂一层石蜡薄膜片, 以创建浅水井, 并在每个井10中放置2到3滴4% 的粉煤灰。适当地给水井贴上标签, 轻轻地将盖子滑倒在粉煤灰填充的油井上。允许细胞在室温下固定20分钟。</li>
	<li>将细胞从一个井移到下一个, 在 DPBS 中洗3次, 5 分钟。</li>
	<li>如果在中性粒细胞活化和固定后不久就不能进行 immunolabelling, 则通过将盖子滑面贴在吸光度纸上, 使盖子滑干。在4摄氏度的标签 12-井培养板上可以储存1周的盖子, 直到进一步分析。使用与4.4 中描述的相同技术, 在继续下一步之前, 在 DPBS 中3次清洗细胞1分钟。</li>
	<li>要 permeabilize 细胞, 使用4.4 中描述的技术, 轻轻地将盖子滑倒在 1% NP-40 的下落上, 1 分钟。在 DPBS 洗3次, 在摇杆上1分钟。</li>
	<li>随机将每个样本分配给一个数字, 并使用菱形点标记相应地标记盖玻片。</li>
	<li>准备和标签的个别培养皿内衬石蜡膜和地方100至 200 ul 5% 山羊血清上的影片 (堵塞缓冲区)。将盖玻片倒在阻塞缓冲区上。放置在摇杆和孵化1小时在37摄氏度。</li>
	<li>在 DPBS 洗3次, 在摇杆上5分钟。</li>
	<li>稀释原抗体 (1:400 只兔多克隆抗瓜组蛋白 H3 (citH3), 5% 只山羊血清)。在标记的培养皿中孵化细胞, 内衬石蜡膜。把每个盖子滑倒在50到 100 ul 的稀释抗体溶液。放置在摇杆和孵化1小时在37°c。</li>
	<li>在 DPBS 洗3次, 在摇杆上5分钟。</li>
	<li>在5% 山羊血清中共轭荧光 (1:200) 的稀释次生抗体。在标记的培养皿中孵化细胞, 内衬石蜡膜。把每个盖子滑倒在50到 100 ul 的稀释抗体溶液。放在摇杆上, 在黑暗中孵化1小时37摄氏度。</li>
	<li>在黑暗中的摇杆上洗3次 DPBS 5 分钟。</li>
	<li>对于髓 (MPO) 的并发检测, 按照 Li等人所描述的修改后的双 immunolabeling 协议执行以下步骤。13
</li>
	<li>在标记的培养皿中孵化细胞, 内衬石蜡膜。将每个盖子滑倒在100到 200 ul 的10% 兔血清在柔和的摇摆下 (隔夜, 4 °c, 在黑暗中)。</li>
	<li>在黑暗中的摇杆上洗3次 DPBS 5 分钟。</li>
	<li>50微克/毫升游离山羊抗兔骨细胞在黑暗中的摇杆在室温下的2小时的孵化单元。</li>
	<li>在黑暗中的摇杆上洗3次 DPBS 5 分钟。</li>
	<li>稀释原发抗体 (1:200 只兔多克隆抗人过氧化物酶抗体) 5% 只山羊血清。在标记的培养皿中孵化细胞, 内衬石蜡膜。把每个盖子滑倒在50到 100 ul 的稀释抗体溶液。在一个摇杆和孵化1小时在37°c 在黑暗中。</li>
	<li>5% 只山羊血清和孵育的稀释二次抗体共轭荧光在4。8</li>
	<li>在黑暗中的摇杆上洗3次 DPBS 5 分钟。</li>
	<li>应在第二个免疫标签步骤中排除与主要抗体的潜伏期, 以制备干扰控制。</li>
	<li>染色的 DNA 与300毫微米 4 ', 6-Diamidion 2-苯基吲哚, 盐酸盐 (DAPI) 5 分钟在黑暗中的室温。</li>
	<li>在黑暗中的摇杆上洗3次 DPBS 1 分钟。</li>
	<li>在玻璃滑梯上应用 antifade mountant 滴 (~ 50 ul)。轻轻地点击盖玻片的边缘在吸水纸上, 以消除多余的缓冲, 然后再安装盖玻片与细胞颠倒。安装介质应在盖玻片和玻璃滑块之间形成薄层。要去除气泡, 用细钳轻轻按压盖玻片。</li>
	<li>允许样品在黑暗中过夜4摄氏度。安装介质应硬化的微观分析与浸入式透镜。储存样品在4°c 在黑暗中。</li>
</ol>5. 中性粒细胞胞外陷阱量化
<ol><li>盲操作者获取和分析显微镜图像的实验条件。</li>
	<li>使用荧光显微镜在40X 放大倍数, 在各自的渠道下, 每盖玻片的不同区域随机抽取10张图像。确保每个通道的曝光时间、亮度和对比度在图像的采集过程中保持一致。</li>
	<li>使用 ImageJ (NIH) 等可用软件分析图像。根据 cfDNA、citH3 和过氧化物酶的共同定位确定网 (图 3A, B)。在每个实验条件下, 量化10个随机场中的网和中性粒细胞数量。释放的网数可以表示为每个实验条件的比率 (网数: 中性粒细胞总数)。</li>
	<li>为了评价治疗对细胞内 citH3 表达的影响, 在各自的通道下, 以40X 倍的比例在10个随机场中计数细胞内 citH3 的中性粒细胞数目和无细胞内 citH3 的中性粒细胞数目 (图 3, 红色箭头)。细胞内 citH3 表达可以表达为中性粒细胞数与胞内 citH3 的比例与无细胞内 citH3 的中性粒细胞数目的比值。</li>
</ol>

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作者没有什么可透露的。

我们在这里提出一个协议, 以观察细胞 permeant 染料和细胞 impermeant 染料的活犬中性粒细胞细胞核构象和 cfDNA 释放的变化。该方法的主要优点是, 它允许在无细胞固定的情况下, 通过高分辨率显微术实时检测网络形成, 为观察体外网状形成提供了一个简单而有价值的工具。由于这种化验不需要抗体检测的净成分, 它是理想的研究 NETosis 在物种特定的抗体有限的可用性。利用这一方法, 作者证明, 犬中性粒细胞在 NETosis13之前, 在 LPS 或 decondensation 的刺激下进行染色质染色。这项技术的一个主要缺点是它不是特定于 NETosis 的。因为细胞 impermeant 染料还会在坏死细胞或从裂解中性粒细胞中释放的核酸中染色核酸, cfDNA 或细胞 impermeant 阳性细胞可能被错误地解释为网或 NETosing 中性白细胞。另外, NETosis 的特异性可以通过包括 PAD4-inhibitor、Cl-amidine 预处理的活性中性粒细胞来增加。我们在这里展示了在整个隔离过程中确保无菌和适当处理中性粒细胞的重要性, 因为污染和纯粹的力量会导致体外坏死和细胞裂解。我们还建议遵守所有议定书的适当温度, 防止长时间 (> 1 分钟) 低渗裂解。为确定孤立中性粒细胞超时工作的可行性, 强烈建议采用台盼蓝排斥试验以及由静态中性粒细胞组成的负控制。
由于细胞不透水核酸染料检测的 cfDNA 不特定于 NETosis, 我们开发了一种双 immunolabeling 协议, 用于定量测定犬中性粒细胞的体外网状形成和胞内 citH3 表达。当中性粒细胞释放像过氧化物酶这样的颗粒蛋白, 连同 cfDNA 和 citH3 进入胞外空间时, 基于 cfDNA、citH3 和过氧化物酶的协同定位的净量化比其他的检测方法, 如 cfDNA、核或 DNA-过氧化物酶复合物更具体。14. 由于与犬蛋白交叉反应的抗体的供应有限, 我们使用了来自另一物种 (兔子) 的主要抗体。为了防止在染色过程的任一步骤中, 通过残留的游离结合部位捕获原抗体, 我们首先利用兔血清, 然后用游离的碎片将游离结合部位饱和。这一程序的一个关键步骤是确保适当的洗涤, 以防止免疫球蛋白和晶圆碎片的过量结合, 可能干扰检测第二种蛋白质的利益。我们还建议测试不同稀释的主要和次要抗体, 以尽量减少非特异性的约束力和干扰。为了确保两个 immunolabelling 步骤中的二级抗体特异地结合其主要抗体, 调查人员应包括在第二个 immunolabelling 步骤中排除两种主要抗体的2种不同的控制。当检测到两次抗体的信号时, 就会发生非特定的绑定。
由酶 peptidylarginine deminase 4 (PAD4) 催化的组蛋白 citrullination 或脱氨作用, 在小鼠和人类15,16的细菌相关 NETosis 中是必不可少的。利用这一化验, 我们能够确定脂多糖诱导的 NETosis 在狗也需要组蛋白 H3 citrullination 的垫13。为了测试感兴趣的兴奋剂是否通过 PAD4 激活来诱发 NETosis, 我们建议使用已知的 PAD4 激活剂 (如 PMA 或钙载体, A2318717) 包含阳性控制。如果中性粒细胞显示阳性对照样品中 citH3 的阴性染色, 则必须对该协议进行修订, 直到产生足够的阳性染色。我们还建议包括由静态中性粒细胞或中性粒细胞组成的生物阴性控制, 由垫抑制剂, 氯 amidine。
使用这种技术可以准确地识别出不同的网结构。这可能是特别困难的样品与广泛的 NETosis, 因为合并的网状结构从多个相邻的中性粒细胞可能导致低估的 NETotic 事件。这项化验结果可能与其他更客观的化验, 如流式细胞术或 ELISA, 尚未证实用于犬中性粒细胞。为避免形成汇合网状结构, 研究者可以将细胞的浓度降低。另外, 减少总孵化时间可以避免网络结构的广泛 NETosis 和合并。虽然这项协议可以用来调查其他物种的 NETosis, 根据作者的经验, 中性粒细胞对 LPS 或 PMA 的反应在物种之间会有很大差异。
狗网的发现凸显了 NETosis 是微生物感染和免疫介导疾病中先天免疫的一种保守机制。这里所提供的化验方法可以作为研究狗和其他物种 NETosis 的宝贵工具。对脓毒症诱导的 NETosis 的更好的理解将有助于进一步研究脓毒症的新治疗策略的发展。

中性粒细胞是短寿命的颗粒, 负责初步防御入侵病原体。嗜中性粒细胞被招募到感染部位, 通过吞噬、脱粒和产生活性氧 (ROS)1消除微生物。在细菌或内毒素存在的情况下, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 由细胞外染色质修饰的组蛋白和颗粒蛋白, 如弹性蛋白酶和髓 (过氧化物酶)2。虽然蚊帐具有不可缺少的抗菌性能, 但越来越多的实验和临床证据表明, 脓毒症的过度过度网络形成可能导致多器官功能障碍和死亡3,4,5,6。
由于蚊帐可能在犬类中起到类似的病理生理作用, 预防或减少网状形成的治疗性干预可以作为脓毒症动物的新治疗策略。因此, 需要一种可靠的技术来评估和量化狗的 NETosis 和网络成分。包括无细胞 DNA (cfDNA) 和核在内的净成分在犬中性粒细胞和7、8、9的临床犬血浆中得到了评价。通过荧光化验, Goggs 和 Letendre 发现, 脓毒症犬的 cfDNA 水平比健康犬高8。虽然这些技术是高度客观和定量的, 测量 cfDNA 和核作为标记的 NETosis 是非特异的, 因为它们可以从坏死细胞以外的 NETosing 中性粒细胞。在这里, 我们描述了一种技术, 利用荧光显微镜检查活 NETosing 中性粒细胞的行为。我们还详细介绍了一种改进的协议, 使用双 immunolabeling 主观量化的网及其组成部分, 如髓内过氧化物酶和 citH3 在犬中性粒细胞10。

<table><tbody><tr><td>Dextran from &lt;em&gt;Leuconostoc&lt;/em&gt; spp.</td><td>Sigma</td><td>31392</td><td>Molecular weight 450,000 &amp;ndash; 650,000</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Life Sciences</td><td>17144002</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>ThermoFisher Scientific</td><td>A1285801</td><td>With divalent cations</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190136</td><td>Without divalent cations</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; O55:B5</td><td>InvivoGen</td><td></td><td></td></tr><tr><td>SYTOX Orange Fluorescent Nucleic Acid Stain</td><td>ThermoFisher Scientific</td><td>S11368</td><td>5 mM in DMSO; stains in cells with permeable membranes</td></tr><tr><td>SYTO Green 16 Fluorescent Nucleic Acid Stain</td><td>ThermoFisher Scientific</td><td>S7578</td><td>1 mM in DMSO; stains in cells with intact membranes</td></tr><tr><td>Surface-Amps NP-40</td><td>Pierce</td><td>28324</td><td></td></tr><tr><td>Poly-D-Lysine coated coverslips</td><td>neuVitro</td><td>H-12-pdl</td><td>12 mm diameter</td></tr><tr><td>Anti-citrullinated histone H3 antibody</td><td>Abcam</td><td>Ab5103</td><td></td></tr><tr><td>Unconjugated goat anti-rabbit Fab fragments</td><td>Jackson ImmunoResearch</td><td>111-007-003</td><td>Specificity: IgG (H+L)</td></tr><tr><td>Anti- Myeloperoxidase antibody</td><td>Dako</td><td>A0398</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylin (DAPI)</td><td>Life Technologies Corporation</td><td>D1306</td><td></td></tr></tbody></table>

in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy

为了应对入侵的病原体, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 它们是用组蛋白和抗菌蛋白修饰的 DNA 胞外网络。脓毒症期间过度的净形成 (NETosis) 和 citH3 释放与小鼠和人类多器官功能障碍和死亡有关, 但其对狗的影响是未知的。本文介绍了一种从全血中分离犬中性粒细胞的方法, 以观察和定量 NETosis。由葡聚糖沉淀产生的富含白细胞的等离子体, 通过商业上可用的密度梯度分离介质和细胞计数和生存能力测试收集的颗粒分离。为了观察实时 NETosis 在活中性粒细胞, 细胞 permeant 和细胞 impermeant 荧光核酸染色添加到中性粒细胞激活, 无论是由脂多糖 (LPS) 或佛波 12-酯 13-醋酸酯 (PMA)。随着时间的推移, 荧光显微镜观察到核形态和网状形成的变化。体外NETosis 进一步的特点是定位的无细胞 DNA (cfDNA), 髓 (过氧化物酶) 和瓜组蛋白 H3 (citH3) 使用改良的双 immunolabelling 协议。为了客观地量化网络形成和 citH3 表达, 使用荧光显微镜, 网和 citH3-positive 细胞是量化的盲目的方式使用可用的软件。该技术是评价犬中性粒细胞体外能力 NETosis 的一种特殊检测方法。

利用活体细胞成像协议, 研究人员可以观察细胞核形态、细胞膜完整性和 cfDNA 在活中性粒细胞中的存在。细胞 impermeant 核染料污渍细胞核酸红色的细胞与受损细胞膜。另一种细胞 permeant 染料, 标签细胞内的核酸在活细胞与完整的等离子膜。所有完整的中性粒细胞, 无论其治疗, 应出现绿色, 并表现出的特点肿块核在0到30分钟后刺激 (图 2A)。经治疗的中性粒细胞的细胞核开始失去其肿块的外观约60分钟, 并继续 decondense, 直到释放 cfDNA (图 2B)。LPS, 一种较慢和更具生理刺激性的犬中性粒细胞, 通常不会导致染色质 decondensation 或 NETosis, 直到90分钟的刺激 (图 2B)。由120分钟, LPS 和活化活性中性粒细胞可以看到周围的 cfDNA。与 LPS 相比, 由于细胞 impermeant 染料染红, 更多的活化中性粒细胞失去了细胞膜的完整性 (图 2C)。静态中性粒细胞 (DPBS 控制) 应保持肿块核和完整的血浆膜在整个潜伏期 (图 2A-C)。
使用双 immunolabeling 协议检测出由 cfDNA、citH3 和过氧化物颗粒组成的网状成分。根据 cfDNA、citH3 和髓过氧化物酶的联合定位, 对 LPS 和 PMA 的反应, 分别显示在图 3A 和 3B中。脂多糖刺激的中性粒细胞为180分钟, 在靠近附近的中性粒细胞中产生离散的网状状支架 (图 3A)。相比之下, 活化的中性粒细胞产生了大量的网, 明显大于面积 (图 3B, D)。酶 peptidylarginine deiminase (垫) 催化的组蛋白 H3 的 Citrullination 是 NETosis 的标志。citH3 是一种有效的垫型兴奋剂, 其结果是, 与静态和 LPS 刺激的中性粒细胞相比, 表达胞内的神经细胞数量更高 (图 3E)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58083/58083fig1.jpg"> 图 1: 演示了犬中性粒细胞分离步骤的示意图.稀释血气全血第一次混合3% 葡聚糖, 以沉淀红细胞 (红细胞)。然后将富含白细胞的等离子体收集并分层到同样数量的商业可用密度梯度分离介质上。经离心 (400 x g, 30 分钟), 提取红细胞粒细胞层, 用低渗裂解去除剩余红细胞。添加1.8% 氯化钠后, 粒细胞被纺下 (112 x g, 5 分钟) 和悬浮在专用缓冲。<a href="https://www.jove.com/files/ftp_upload/58083/58083fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58083/58083fig2.jpg"> 图 2: 活体犬中性粒细胞的典型荧光图像.分离的中性粒细胞以100µg/毫升脂多糖, 100 nM, 或 Dubecco 磷酸盐缓冲盐水 (DPBS) 的当量体积为负控制, 共180分钟, 活完好的中性粒细胞中的核酸染色绿色, 使用细胞 permeant染料, 而 cfDNA 和中性粒细胞与受损细胞膜被染红的细胞 impermeant 染料。(A、D)DPBS 和 LPS 治疗的中性粒细胞都维持一个完整的细胞膜与肿块核 (星号)。(A、E)活性中性粒细胞的细胞核开始接受 decondensation (箭头)。(B、F)由90分钟, 大多数细胞核在 LPS 或活化激活中性粒细胞已经失去了他们的肿块外观 (蓝色箭头)。(C) 120 分钟后, 所有的活化中性粒细胞都有渗透性的血浆膜, 周围有 cfDNA, 而 cfDNA 可以看到周围少量的 LPS 活化中性粒细胞 (箭头)。静态中性粒细胞并没有产生 cfDNA, 也没有在任何时间点 (A-C) 破坏细胞膜。(A-B)原始40X 放大倍数。刻度条 = 100 µm (D F) 80X 放大倍数。缩放条 = 30 µm. 实验被复制了两次从中性粒细胞分离从3不同的捐赠者。<a href="https://www.jove.com/files/ftp_upload/58083/58083fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58083/58083fig3.jpg"> 图 3: NET 的形成.(丙)体外网状形成的具有代表性的免疫荧光图像。用 (a) 100 citH3/毫升 LPS, (B) 100 nM 或 (C), 从单个捐献者中分离出来的中性粒细胞被固定, 透和染色 DNA (蓝色), 髓 (过氧化物酶, 绿色) 和瓜组蛋白 H3 (µg, 红色) DPBS在 (A) LPS 和 (B) cfDNA 活化的中性粒细胞中, 对180分钟网进行了概述 (点线)。用红色箭头表示 citH3 细胞内表达的例子。(C) 静态中性粒细胞内不存在网状或胞内 citH3。刻度条 = 100 µm (D, E) 有代表性的盒子和净形成的晶须图和细胞内 citH3 在4条狗的中性粒细胞中的表达。该框从25到 75的百分点和晶须跨度从最小到最大值。LPS 和 PMA 刺激导致显著 (D) 净形成和 (E) 胞内 citH3 表达比静态中性粒细胞。+ 表示平均值。<a href="https://www.jove.com/files/ftp_upload/58083/58083fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

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

我们描述了从全血中分离犬中性粒细胞的方法, 用荧光显微镜观察活中性粒细胞的网状形成。还描述了使用免疫荧光显微镜定量分析网络形成和瓜组蛋白 H3 (citH3) 表达的协议。

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

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

8.7K 次观看.  University of California, Davis. 我们描述了从全血中分离犬中性粒细胞的方法, 用荧光显微镜观察活中性粒细胞的网状形成。还描述了使用免疫荧光显微镜定量分析网络形成和瓜组蛋白 H3 (citH3) 表达的协议。

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

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

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

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. ...

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. ...

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 ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

医学

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 ...

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

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嗜中性粒细胞胞外的陷阱：如何生成和可视化

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

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

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

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

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

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