Name: neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment
Uploaded: 2018-08-03T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

我们感谢 j. 筱女士和我 Nieda 的技术和文书工作。此外, 我们感谢 Drs, Hidenori Haruta, 宪太郎 Kurashina 和葛屋高桥为他们合作的样品采集在手术室。这项工作得到了日本教育、科学、体育和文化部和日本促进科学学会 (17K10606) 的科学研究资助。

LDN 是由接受本研究的病人获得的, 并获 Jichi 医科大学机构评审委员会批准。
1. 腹腔 Lavages LDN 的分离和净检测
<ol><li>样品采集<ol>
<li>将1000毫升的正常无菌生理盐水直接注入腹腔内, 经胃肠道恶性肿瘤腹部手术的病人在伤口闭合前, 将其注射至腹腔内。 注: 样本是从病人接受胃切除, 结肠切除, 或食管切除没有偏见的基础上的年龄或性别。盐水被转移到一个容器里, 一分钟内倒入整个腹部。这通常是作为术后腹膜冲洗没有明显的影响病人。</li>
		<li>广泛冲洗腹腔, 至少1分钟。 注: 建议将输液液慢慢地与外科医生的手搅拌, 以使样品均匀。</li>
		<li>用四50毫升注射器恢复200毫升灌洗液。 注: 有时用橡胶连接器来取流体。</li>
	</ol>
</li>
	<li>用粒细胞特异性单克隆 CD66b16对腹膜 LDN 进行纯化。 注: 由于密度梯度离心后的中间层含有许多单个核细胞, CD66b 单克隆抗体进行了阳性变形。<ol>
<li>将腹腔灌洗液转移至50毫升管。 注: 在这一步, 通过100µm 尼龙过滤器的液体, 以消除杂质。</li>
		<li>以 270 x g 为7分钟, 在 RT 上离心腹膜液。</li>
		<li>并用重悬5毫升的 PBS 中的小球, 0.02% EDTA。</li>
		<li>仔细覆盖5毫升的细胞悬浮在3毫升密度梯度解决方案。</li>
		<li>离心机在 1700 x g 的15分钟, 在 RT 没有任何休息。</li>
		<li>使用吸管将含有中间层的 2-3 毫升溶液 (图 1A) 收获, 并将其与10毫升的 PBS 混合使用 0.02% EDTA。</li>
		<li>将腹膜液以 400 x g 为7分钟在 RT 上离心, 然后丢弃上清。</li>
		<li>再添加10毫升的 PBS, 0.02% EDTA, 离心机在 270 x g 在 RT 的7分钟. 弃上清。</li>
		<li>将细胞颗粒 (1 x 107) 溶解在60µL 的缓冲器中, 用于磁性细胞分离试剂盒。</li>
		<li>添加20µL 的 Fc 块到小球和孵化10分钟在4摄氏度。</li>
		<li>添加20µL 的 anti-CD66b 共轭微球和孵化的额外10分钟4摄氏度。</li>
		<li>在500µL 的 mac 缓冲器中清洗并重新悬浮小球。</li>
		<li>将细胞悬浮物应用于合适的磁选机磁场中的磁柱上, 通过用15毫升的缓冲器洗涤柱, 收集含有 CD66b (-) 细胞的流动。</li>
		<li>从磁选机中取出该列, 然后将柱塞牢固地推入柱形, 立即冲洗出磁标记的 CD66b (+) 单元格。 注: CD66b (+) 细胞的数量主要由流化分析确定的中性粒细胞组成, 这表明 > 95% 的 CD66 (+) 分数对 CD11b、CD15 和 CD16 是阳性的, 但对 CD14 是阴性的。</li>
	</ol>
</li>
	<li>世代网<ol>
<li>离心后在 270 x g 7 分钟的 RT, 重新暂停隔离 LDN (5 x 106) 在1毫升的 RPMI1640 与10% 的 FCS。</li>
		<li>在 5% CO2的37˚C 中, 在6井聚 l-赖氨酸涂层板 (直径6厘米) 上培养 LDN 2 小时。</li>
		<li>添加绿色荧光 counterstain (膜不透水染料染色的细胞核和染色体) 在最终浓度的5µM。</li>
		<li>立即观察网的形态 (在荧光显微镜下从 LDN 中排出的胞外 DNA 成分)。</li>
	</ol>
</li>
</ol>2. 用红荧光细胞链接染料染色肿瘤细胞
<ol><li>准备人类癌细胞系 MKN45, NUGC 和 OCUM-1。</li>
	<li>1 x 107细胞与 PBS + 0.02% EDTA 在15毫升管和离心机在 270 x g 为7分钟在 RT。</li>
	<li>添加1毫升的溶液染料染色的小球, 和吸管轻轻。</li>
	<li>将4µL 的红色荧光细胞链接剂染料溶于1毫升溶液中染色, 并与步骤2.2 中的细胞悬浮液混合。</li>
	<li>在 RT 中孵化4分钟的小球。</li>
	<li>添加4毫升的 DMEM (10% 的血清), 以阻止染色反应。</li>
	<li>离心机 270 x g 7 分钟, 并丢弃上清。</li>
	<li>重复步骤2.6 和2.7 两次。</li>
	<li>检查与荧光显微镜 (励磁 = 551 毫微米, 发射 = 567 毫微米) 那肿瘤细胞被染红。</li>
</ol>3. 肿瘤细胞对蚊帐黏附力的分析
<ol><li>再次暂停红色荧光染色肿瘤细胞 (1 x 106) 在1毫升的 RPMI 1640 补充与 0.1% BSA。</li>
	<li>将肿瘤细胞添加到生成网的 LDN 培养中, 如步骤1.3 所述。</li>
	<li>孵育5分钟, 在37˚C, 使肿瘤细胞接触网。 注: 长时间孵化诱导肿瘤细胞粘附到 LDN 或板上。</li>
	<li>通过添加2毫升的 prewarmed 介质 (0.1% BSA + RPMI 1640) 并旋转盘子, 去除介质并轻轻地冲洗水井。</li>
	<li>重复步骤3.4 中的洗涤过程。 注: 由于网弱附着在板材上, 洗涤应尽可能轻柔地进行, 以避免网本身的去除。</li>
	<li>添加绿色荧光染料染色的细胞核和染色体的最终浓度为5µg/毫升, 以可视化的网。</li>
	<li>观察网和附上肿瘤细胞使用适当的过滤器 (绿色, 励磁 = 504 毫微米, 发射 = 523 毫微米; 红色, 励磁 = 551 毫微米, 发射 = 567 毫微米)。</li>
	<li>合并这些数字, 以显示被网困住的肿瘤细胞。 注: 在一些实验中, DNA 降解酶添加到 LDN 培养 (100 U/毫升的最终浓度) 5 分钟前共孵化5分钟。</li>
</ol>4. 被困肿瘤细胞的时间推移视频分析
<ol><li>在35毫米的圆形盘子中培养腹膜 LDN, 如步骤1.3 所述。</li>
	<li>添加绿色荧光染料染色的细胞核和染色体, 以可视化网如步骤1.3 所述。</li>
	<li>孵化1分钟后, 取出培养基, 加入2毫升 0.1% BSA + RPMI 1640。</li>
	<li>删除介质并添加无瑕 1 x 106 MKN45 细胞悬浮在 0.1% BSA + RPMI 1640。在 RT 孵化5分钟。</li>
	<li>如步骤3.4 中所述, 通过轻轻冲洗去除任何未连接的肿瘤细胞, 并添加 DMEM 与 10% FCS 补充100毫升青霉素和100µg/毫升链霉素。</li>
	<li>在全视图细胞观察系统中安装培养皿, 并选择适当的领域, 其中肿瘤细胞被网困。</li>
	<li>继续共培养2天, 并采取连续照片的领域每6分钟的正常光和荧光, 分别检测 MKN45 细胞和网。</li>
	<li>在每个 timepoint 上叠加图像, 并使用图像查看器软件构造延时视频。</li>
</ol>

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作者声明他们没有竞争的财政利益。

以前的研究报告说, 循环肿瘤细胞可以被网络基质所困在体内10,11。转移性乳腺癌细胞已被证明是刺激中性粒细胞和诱导形成的蚊帐, 这有助于肿瘤细胞生长的目标器官17。此外, 我们发现, 从术后灌洗液中 LDN 的短期培养可以有效地诱捕肿瘤细胞, 而无需进一步刺激15。这些观察表明, 在转移过程中, 网的机械参与?...

循环血中变形核中性粒细胞通常通过密度梯度制备方法与单个核干细胞分离。然而, 一些被称为低密度中性粒细胞的中性粒细胞 (LDN), CD11b (+), CD15 (+), CD16 (+) 和 CD14 (-) 表型, 与单个核细胞共纯化。LDN 的相对数量在各种病理条件下显著增加, 包括自身免疫性疾病1、2、脓毒症3和癌症4、5。以前的研究表明, LDN 是一种表型和功能上明显的中性粒细胞6。应注意的是, 循环血液中的 LDN 比正常密度的中性粒细胞2,7更容易产生中性粒细胞胞外陷阱 (蚊帐)。网是由核酸、组蛋白、蛋白酶、颗粒和胞浆蛋白组成的网状结构, 可以有效地诱捕和破坏病原体8。
最近, 蚊帐已被证明不仅捕获微生物, 而且还有血小板和循环肿瘤细胞, 可以帮助血栓形成9和肿瘤转移10,11。然而, 网络与血小板或肿瘤细胞之间的粘接作用背后的分子机制还不清楚。最近,体外黏附检测发现髓细胞白血病 (K56212) 和肺癌细胞 (A54913) 附着在网通过β1和β3整合。作者利用从中性粒细胞中分离的净存量, 用佛波 12-酯 13-醋酸酯 (PMA) 活化的网状物作为粘附基底14。虽然这种检测可以检测到在无中性粒细胞的情况下与净成分的实际相互作用, 但是, 高速离心分离的 "无细胞网" 是否保留了与所生产的网相同的分子结构体内。最近, 我们发现腹部手术后腹腔灌洗液含有许多成熟的 LDN, 产生大量的网状物, 附着于肿瘤细胞引起的腹膜转移15。在这项研究中, 我们成功地检查了肿瘤细胞对完整网的黏附力, 没有任何物理操作。在这里, 我们展示了一种检测网络与游离肿瘤细胞之间粘接作用的技术的细节。

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare, SWEDEN</td><td>17-1440-02</td><td></td></tr><tr><td>StraightFrom&amp;trade; Whole Blood CD66b MicroBeads</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-104-913</td><td></td></tr><tr><td>Fc block</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-059-901</td><td></td></tr><tr><td>MACS Rinsing Solution</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-091-222</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-091-376</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-041-306</td><td></td></tr><tr><td>MACS Magnetic Separator</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-042-501</td><td></td></tr><tr><td>SYTOX green nucleic acid stain 5mM solution in DMSO</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>S7020</td><td></td></tr><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>P9691</td><td></td></tr><tr><td>Diluent C</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>CGLDIL</td><td></td></tr><tr><td>RPMI1640 Medium</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>R8758</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium-high glucose (DMEM)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>D5796</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>D8537</td><td></td></tr><tr><td>0.5mol/l-EDTA Solution (pH 8.0)</td><td>nacalai tesque, Japan</td><td>06894-14</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, USDA-approved regions</td><td>gibco by life technologies, Mexico</td><td>10437-028</td><td></td></tr><tr><td>Bovine Serum Albumin lyophilized powder, &amp;ge;96% (agarose gel electrophoresis)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>A2153</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies Japan</td><td>15140-122</td><td></td></tr><tr><td>Plasmocin Prophylactic</td><td>InvivoGen, San Diego, CA-USA</td><td>ant-mpp</td><td></td></tr><tr><td>DNase I</td><td>Worthington, Lakewood NJ)</td><td>LS002138</td><td></td></tr><tr><td>Poly-L-Lysine-Coated MICROPLATE 6Well</td><td>IWAKI, Japan</td><td>4810-040</td><td></td></tr><tr><td>Poly-L-Lysine-Coated MICROPLATE 24Well</td><td>IWAKI, Japan</td><td>4820-040</td><td></td></tr><tr><td>fluorescein stereomicroscope</td><td>BX8000, Keyence, Osaka Japan</td><td>BZ-X710</td><td></td></tr><tr><td>Whole view cell observation system</td><td>Nikon, Kanagawa, Japan</td><td>BioStudio (BS-M10)</td><td></td></tr><tr><td>MKN45 human gastric cancer cell line</td><td>Riken, Tukuba Japan</td><td>N/A</td><td></td></tr><tr><td>NUGC-4 human gastric cancer cell line</td><td>Riken, Tukuba Japan</td><td>N/A</td><td></td></tr><tr><td>OCUM-1 human gastric cancer cell line</td><td>Osaka City University, Japan</td><td>N/A</td><td>Gift from Dr. M.Yashiro</td></tr></tbody></table>

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

活化中性粒细胞释放中性粒细胞胞外陷阱 (网), 可以捕获和破坏微生物。最近的研究表明蚊帐参与了各种疾病过程, 如自身免疫性疾病、血栓形成和肿瘤转移。在这里, 我们展示了一个详细的体外技术, 以检测在游离肿瘤细胞的诱捕过程中的净活动, 它生长后, 附着在网。首先, 我们收集了 laparotomies 患者术后腹腔灌洗液中的低密度中性粒细胞 (LDN)。LDN 的短期培养导致了大量的网状形成, 可见绿色荧光核和染色体 counterstain。在人类胃癌细胞系 MKN45、OCUM-1 和 NUGC-4 与网的共同孵化后, 许多肿瘤细胞被网所困住。随后, 由于网 DNase 的降解, 附着物完全被取消了. 时间推移视频显示, 网陷的肿瘤细胞没有死亡, 而是在持续的文化中大力生长。这些方法可用于检测网与各种类型的细胞和材料之间的粘接作用。

在2小时的文化中, 从腹膜灌洗液中提取的 CD66b (+) LDN 显示了用绿色荧光染料染色核和染色体的弦结构 (图 1B), 而 CD66b (-) 单个核细胞没有 (图 1C)。然而, 当 LDN 培养的 100 U/毫升 DNase I, 特征结构被破坏 (图 1D), 表明它们是由细胞外 DNA 排出的中性粒细胞。当 LDN 在没有涂层的塑料板上进行培养时, 大量的网状簇?...

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

在这里, 我们提出一种方法, 其中人类低密度中性粒细胞 (LDN), 从术后腹腔灌洗液中恢复, 产生大量的中性粒细胞外圈闭 (网), 并有效地诱捕后生长的游离肿瘤细胞。

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

neutrophil-extracellular-traps-generated-low-density-neutrophils

Research

JoVE Journal

Cancer Research

8.7K 次观看.  Jichi Medical University. 在这里, 我们提出一种方法, 其中人类低密度中性粒细胞 (LDN), 从术后腹腔灌洗液中恢复, 产生大量的中性粒细胞外圈闭 (网), 并有效地诱捕后生长的游离肿瘤细胞。

视频: Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

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

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

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

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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.

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

摘要

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嗜中性粒细胞胞外的陷阱：如何生成和可视化

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

隔离和中性粒细胞的表征与抗肿瘤性质

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

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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