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, ...

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 Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392</td>
			<td>Molecular weight 450,000 &#8211; 650,000</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Life Sciences</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>A1285801</td>
			<td>With divalent cations</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190136</td>
			<td>Without divalent cations</td>
		</tr>
		<tr>
			<td>E. coli 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?...

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 ...

in-vitro-canine-neutrophil-extracellular-trap-formation-dynamic

Research

JoVE Journal

Immunology and Infection

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

8.6K 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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

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. ...