我们感谢 Suchitra Swaminathan 博士的技术援助, 他是西北范伯格医学院流式细胞术的核心。资助是由 (NIH) DK101675 提供的。

所有的动物工作都是由西北 IACUC 批准的。所有的实验都是按照所有相关的规章制度和制度准则完成的。对于献血者, 知情同意书被提出并签署;此外, 这项研究中的所有人类学科都是按照《机构和联邦人类福利准则》处理的。
注意: 协议步骤列在以下小节中: (i) 小鼠骨髓细胞分离;(ii) 中性粒细胞与小鼠骨髓隔绝;(iii) 中性粒细胞与人血隔绝;(四) MP 与中性粒细胞上清液 (由离心) 隔离;(五) 用西方印迹描述 MPs;(vi) 流式细胞术对 MPs 的表征;(viii) 孤立的 MPs 在伤口愈合研究中的应用
1. 小鼠骨髓细胞分离
<ol style="margin-left: 40px;"><li>
小鼠后腿的股骨和胫骨解剖
	<ol>
<li>准备一个安乐死盒 (例如, 一个空的1毫升提示盒) 吸入麻醉。添加几滴100% 异氟醚到一个不育布贴在塑料盒盖内。根据新的 IACUC 准则, 动物不应该直接接触异氟醚, 因此在老鼠和含有异氟醚的纱布之间放置一个尖端支架。 注: 异氟醚是一种强大的吸入麻醉药, 应在通风罩内极端小心处理。</li>
		<li>弄死小鼠按 IACUC 推荐。将鼠标放在步骤1.1.1 中描述的安乐死框中, 1–5分钟, 直到它停止呼吸。通过执行颈椎脱位来确保动物的死亡。</li>
		<li>用不锈钢、12厘米弯曲、0.17 毫米 X 0.1 毫米镊子和在上肢和下肢之间切开皮肤的腹部皮肤。将皮肤从这里剥离到老鼠身体的最远端部分, 包括下肢。用10厘米长, 直解剖剪刀, 从后腿移除肌肉和脱臼髋部关节留下股骨头完整。</li>
		<li>用剪刀解剖股骨和胫骨的肌肉, 并将股骨与胫骨分开。确保骨骼的两端保持完好, 因为它们含有大量的骨髓。</li>
		<li>通过在纸巾内滚动骨头来除去所有剩余的肉。将干净的骨骼放在含有无血清培养基的培养皿中 (可以持续使用,即, Dulbecco 的改良鹰的培养基 (DMEM), 不含血清或抗生素)。</li>
		<li>在70% 乙醇中冲洗骨头, 然后在森林中。</li>
		<li>在50毫升圆锥管中准备50毫升的森林能持续。将10毫升的可持续森林可持续性转化为10毫升注射器, 并将其附加到25和5/8 英寸口径的针上。</li>
		<li>用剪刀修剪骨头的末端, 直到骨髓的开口 (红颜色) 可见。</li>
		<li>将骨髓细胞冲洗成新的50毫升管。使用10毫升注射器与26和5/8 英寸测量针与大约3毫升/骨的可以持续森林。冲洗完后, 用吹打 (使用一次性1毫升塑料小贴士) 并用重悬管中的细胞, 以打破细胞团聚体。</li>
	</ol>
</li>
</ol>2. 小鼠骨髓中性粒细胞的分离
<ol><li>
红细胞裂解
	<ol>
<li>用离心在 350 x g上将收集到的骨髓颗粒在4摄氏度8分钟。</li>
		<li>并用重悬细胞颗粒在20毫升0.2% 氯化钠约20-30 秒溶解红细胞分数。不要超过三十年代, 因为这将导致中性粒细胞裂解。添加20毫升1.6% 氯化钠, 以恢复渗透。 注意: 这一步可能导致很少的中性粒细胞活化。</li>
		<li>用2毫升的 PBS 和离心机清洗细胞, 2.1.1 以上步骤。移除上清。</li>
		<li>进行密度梯度离心分离 PMNs。</li>
	</ol>
</li>
	<li>
密度梯度离心法分离中性粒细胞
	<ol>
<li>温暖的聚蔗糖和钠葡溶液, 密度为1.119 克/毫升和1.077 克/毫升 (参见材料表), 以室温 (RT) 使用前。</li>
		<li>准备聚蔗糖和钠葡梯度新鲜, 立即在骨髓细胞应用之前, 慢慢地分层3毫升的1.077 克/毫升密度溶液3毫升的1.119 克/毫升密度溶液在15毫升圆锥管。 注: 梯度溶液的制备在很大程度上会导致混合层和不良的中性粒细胞纯度和恢复。</li>
		<li>并用重悬骨髓细胞颗粒在1毫升冰冷 PBS 和叠加产生的细胞悬浮在聚蔗糖和钠葡梯度 (缓慢, 以避免混合层)。</li>
		<li>离心机为30分钟在 850 x g 在25°c 在 RT,没有刹车。将试剂保存在 RT 中, 有效地将中性粒细胞与骨髓中的其他细胞分离。将离心机设置为慢速减速, 以便在离心步骤后有效地形成和保持梯度。</li>
		<li>在 PBS 和1.077 克/毫升密度溶液 (上层) 的界面上视觉定位单个核细胞层。去掉这个层和其余的密度解决方案, 而不干扰中性粒细胞带。</li>
		<li>收集一个完整的中性粒细胞层和一些基础聚蔗糖和钠葡1.119 克/毫升溶液成15毫升管使用转移吸管。 注意: 隔离 PMNs 的纯度将取决于对上层层的完全去除。</li>
		<li>管与过滤 PBS (过滤通过0.1 µm 孔径过滤器) 和离心机在 300 x g 5 分钟, 在4°c。</li>
		<li>使用离心条件, 如步骤 2.2.7, 用2毫升的 PBS 洗涤颗粒 PMNs。</li>
		<li>并用重悬在1毫升的过滤 PBS 和计数的孤立 PMNs 由 hemocytometer。 注: 对于这种隔离方法, 所产生的中性粒细胞纯度为 ~ 85-90%, 这是由流式检测仪评估的。其余的污染分数主要由单核淋巴细胞组成。</li>
	</ol>
</li>
	<li>
中性粒细胞刺激诱导 MP 释放 注: PMNs 可刺激释放 MPs 使用各种活化条件, 包括 n-甲酰基-l-methionyl-leucyl-l-苯丙氨酸 (fMLF), phorbol-12-myristate-13-acetate (PMA), 或干扰素伽玛 (IFNγ)。重要的是, 在模拟之前, PMNs 应该一直放在冰上。<ol>
<li>颗粒 PMNs (300 x 克, 5 分钟, 4 °c) 和并用重悬在0.1 µm 过滤了汉克的平衡盐溶液 (HBSS) 溶液中, 含有刺激剂的选择。为优化刺激, 使用100µL 刺激溶液每 1000万 PMNs 20-30 分钟在37°c 在15毫升管。 注: 在我们以前的工作中成功地使用了以下活化剂浓度: fMLF (0.5-1 µM 为人和2-5 µM 为小鼠 PMNs)、PMA (200 毫微米) 和 IFNγ (100 ng/毫升)。如果需要预先标记的 MPs 的发行, 孵化静态 PMNs (1-5 x 106) 与 n-(2-氨基乙基) maleimide FITC (参见材料表) (1 µM 在100µL HBSS, 15 分钟, 37 °c)。继续执行步骤 2.3. 1-2. 3.3。</li>
		<li>在4摄氏度处旋转 850 x g 5 分钟, 去除 PMNs 并将无细胞上清液转移到新的1.5 毫升离心管中。</li>
		<li>对于从无单元格上清液的 MP 隔离, 请继续执行步骤4.2。</li>
	</ol>
</li>
</ol>3. 中性粒细胞与人血分离
<ol><li>
根据机构内部评级准则, 招募健康志愿者进行血液提取。
	<ol>
<li>从一个健康的志愿者的前臂收集血液到商业上可利用的5毫升含柠檬酸钠的管 (请参阅材料表)。</li>
		<li>吸管5毫升葡聚糖和钠葡溶液 (密度为1.113 克/毫升, 见材料表) 成无菌15毫升管和缓慢层5毫升新鲜隔离的人外周血在它上面。</li>
		<li>离心管50分钟在 400 x g 在 RT (25 °c) 与低加速度和没有刹车。</li>
		<li>在离心结束时, 使用无菌的塑料吸吸管去除血浆和单个核细胞 (上层)。</li>
		<li>将底层 (PMNs) 转移到新的无菌50毫升圆锥管中。</li>
		<li>在 PMNs 中加入等量的0.45% 氯化钠 (混合好, 轻轻旋转管)。</li>
		<li>离心机为10分钟在 400 x g 在25°c。</li>
	</ol>
</li>
	<li>
红细胞裂解
	<ol>
<li>在四十五年代加入10毫升的冰冷, 不育水给颗粒细胞, 其次是10毫升的冰冷无菌 1.8% NaCl, 以恢复渗透。 注意: 应在冰上执行所有的中性粒细胞隔离步骤, 以避免中性粒细胞活化和早产 MP 释放。裂解步骤本身会导致轻微但不显著的中性粒细胞活化, 然而, 它是必不可少的隔离纯中性粒细胞的功能化验。</li>
		<li>离心的细胞为10分钟在 250 x g 在4°c 与软减速。</li>
		<li>重复步骤3.2.1 和3.2.2。</li>
		<li>并用重悬 PMNs 5 毫升冰冷 HBSS, 无钙或镁。计数总活细胞 (使用 hemocytometer 和生存能力染色在1:2 稀释, 参见材料目录) 在刺激之前。 注: 隔离 PMNs 应用于刺激或其他实验2小时内隔离, 以避免功能和细胞变化和细胞死亡。</li>
	</ol>
</li>
</ol>4. MP 与活化中性粒细胞分离上清液
注: 类似的协议用于隔离小鼠和人类 PMNs 的 MPs。
<ol><li>颗粒 PMNs (300 x g, 5 分钟, 4 °c) 和并用重悬在0.1 µm 过滤 HBSS 解决方案, 包含刺激剂的选择 (例如1µm fMLF, 见注: 2.3.1)。为最佳的刺激使用100µL 刺激解答每 1000万 PMNs 20-30 分钟在37°c 在15毫升管子。</li>
	<li>刺激后, 在4摄氏度的 600 x g 处旋转激活 PMNs 5 分钟, 并将无细胞上清液转移到新的1.5 毫升离心管。</li>
	<li>离心机的中性粒细胞清除清液 1.3万 x g, 10 分钟, 4 °c, 以去除电池碎片和转移清除上清到一个新的 ultracentrifuge 管。</li>
	<li>离心机为1小时在 10万 x g, 在4°c。在 MP 存储之前删除上清液。根据需要, 储存颗粒状 MPs 在石蜡密封 ultracentrifuge 管在-80 °c, 直到使用的实验。</li>
</ol>5. 用西方印迹检查中性粒细胞-MPs
<ol><li>添加 1% SDS 缓冲器 (50-200 µL 100 毫米三 pH: 7.4) 到 MPs 颗粒 (从步骤 4.4), 转移到新的1.5 毫升离心管和煮沸裂解 MPs 5 分钟。 注意: 必须调整和优化每个感兴趣的蛋白质的 MPs 和溶解缓冲容积的数量。</li>
	<li>
载入含有10% β-巯基乙醇的负载缓冲器中每车道的中性粒细胞 MPs。 注意: 用我们的方法, 我们加载从相同数量的受刺激 PMNs 隔离的 MPs。<ol>
<li>在10% 聚丙烯酰胺凝胶上分离裂解物 (使用 50-100 V 1-1. 5 小时), 并将总蛋白转移到硝化纤维膜上, 如下所述 9.</li>
	</ol>
</li>
	<li>在 0.05% Tween-20 三缓冲盐水中用5% 非脂牛奶块膜1小时, 然后用适当的初级 (隔夜在4摄氏度) 孵化, 其次是二次酶共轭抗体。</li>
	<li>通过将 chemoluminescence 溶液添加到薄膜中, 并在防光盒 (1-24 小时) 内暴露在 X 射线胶片上, 可视化带。</li>
</ol>6. 流式细胞仪检查中性粒度 MPs
<ol><li>
抗体染色, 适当稀释抗体在外地资产管制署缓冲区 (PBS 与0.1 毫米 EDTA, 0.1% 叠氮化钠)。并用重悬颗粒中性粒细胞-MPs (从 1-3 x10 6 PMNs/条件) 在100µL 抗体溶液中获得。
	<ol>
<li>如果染色的蛋白 v, 并用重悬颗粒中性粒细胞-MPs 在蛋白 V 缓冲器含有5µL FITC 共轭蛋白 v。如果与抗体共染色, 添加所需的抗体 (例如, 请参阅材料表) 直接到蛋白 V 解决方案。添加荧光脂染料, n-(2-氨基乙基) maleimide (见材料表), 1 µM 浓度在100µL 的外地资产管制署缓冲区, 以标签和识别 MPs。</li>
	</ol>
</li>
	<li>在染色液中孵育 MPs 至少20分钟, 在4摄氏度。</li>
	<li>用离心 (1 小时, 10万 x g, 4 摄氏度) 清洗 MPs;放弃上清。</li>
	<li>并用重悬在外地资产管制系统缓冲区中, 并在指定的流式细胞仪上运行样本, 配备升级后的电子设备以提高灵敏度 (请参阅材料表)。</li>
</ol>7. 孤立中性粒细胞-MPs 在创面愈合中研究中性粒细胞功能的应用
<ol><li>
在体内管理中性粒细胞-MPs 到结肠伤口
	<ol>
<li>麻醉小鼠腹腔注射氯胺酮 (100 毫克/千克) 和甲苯噻嗪 (5 毫克/千克) 的混合物。通过踏板反射 (牢固的脚趾捏) 确认麻醉, 必要时进行调整。</li>
		<li>将鼠标放在它的胃上, 使用28厘米的活检钳和一个装有高分辨率照相机的内窥镜 (如用于小动物使用的兽医内窥镜, 请参阅材料表), 沿背侧产生3-5 处浅层伤口。结肠.受伤后, 将小鼠送回置于温度控制 (37 摄氏度) 垫子上的笼子, 直到恢复。</li>
		<li>麻醉小鼠 (如步 7.1.1)。使用内窥镜获取受伤后24小时 (1 天) 造成的伤口图像。让老鼠在步骤7.1.2 中恢复。</li>
		<li>同时 (24 小时后受伤), 管理小鼠中性粒细胞-MPs 从 2 x 106 PMNs 100 µL HBSS + 直接进入每个伤口部位使用基于结肠镜检查的显微注射系统 (使用29克针)9。三天后 (4 天受伤后) 麻醉小鼠 (如步 7.1.1), 并使用内窥镜重新获取愈合伤口的图像。让老鼠在步骤7.1.2 中恢复。</li>
		<li>使用首选的图像分析软件 (请参阅材料表), 在获得的图像中勾勒出可见的伤口区域, 测量在1天和4创伤后的同一伤口面积, 并计算伤口闭合的百分比。</li>
	</ol>
</li>
	<li>
人上皮细胞体外创面愈合
	<ol>
<li>计数 Caco-2 BBe 或 T84, 人肠道上皮细胞 hemocytometer 和种子 5 x 105细胞/以及24井培养板。在标准孵化室的37°c 和 5% CO2孵化细胞。Caco-2 BBe 或 T84 在 48 h9内达到融合。要培养上皮细胞, 请使用 DMEM 和 DMEM-F12 (50:50), 如前所述的9。</li>
		<li>使用吸管尖端和低吸力在单层中产生机械刮伤, 如前所述4,9。使用反相对比度差动显微镜 (使用5X 或10X 目标), 在划伤后立即获取伤口区域的图像, 用作时间 = 0 参考点。</li>
		<li>添加 MPs (从 2-4 X 106 PMNs) 到划伤的上皮细胞单分子膜, 再孵育 24 h (48 h 后伤后, 37 摄氏度与 5% CO2)。此时重新获取伤痕的影像。</li>
		<li>使用首选的图像分析软件 (请参阅材料表), 以测量 t = 0 和 t = 48 h 的伤口面积. 通过确定所选时间点上伤口区域的差异来计算伤口闭合的百分比。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hickey, M. J., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+immunity:+the+host-pathogen+encounter+in+blood+vessels.">Intravascular immunity: the host-pathogen encounter in blood vessels.</a> Nat Rev Immunol. 9 (5), 364-375 (2009).</li><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nat Rev Immunol. 6 (3), 173-182 (2006).</li><li>Kolaczkowska, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+and+function+in+health+and+inflammation.">Neutrophil recruitment and function in health and inflammation.</a> Nat Rev Immunol. 13 (3), 159-175 (2013).</li><li>Butin-Israeli, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+of+microparticles+by+neutrophils+onto+inflamed+epithelium:+a+new+mechanism+to+disrupt+epithelial+intercellular+adhesions+and+promote+transepithelial+migration.">Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration.</a> FASEB J. , (2016).</li><li>Gasser, O., Schifferli, 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=Activated+polymorphonuclear+neutrophils+disseminate+anti-inflammatory+microparticles+by+ectocytosis.">Activated polymorphonuclear neutrophils disseminate anti-inflammatory microparticles by ectocytosis.</a> Blood. 104 (8), 2543-2548 (2004).</li><li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+purification+and+labeling+of+mouse+bone+marrow+neutrophils+for+functional+studies+and+adoptive+transfer+experiments.">Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments.</a> J Vis Exp. (77), e50586 (2013).</li><li>Shet, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+blood+microparticles:+technical+aspects+and+challenges.">Characterizing blood microparticles: technical aspects and challenges.</a> Vasc Health Risk Manag. 4 (4), 769-774 (2008).</li><li>Dey-Hazra, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+microparticles+by+flow+cytometry:+influence+of+centrifugation,+filtration+of+buffer,+and+freezing.">Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing.</a> Vasc Health Risk Manag. 6, 1125-1133 (2010).</li><li>Slater, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Microparticles+Deliver+Active+Myeloperoxidase+to+Injured+Mucosa+To+Inhibit+Epithelial+Wound+Healing.">Neutrophil Microparticles Deliver Active Myeloperoxidase to Injured Mucosa To Inhibit Epithelial Wound Healing.</a> J Immunol. 198 (7), 2886-2897 (2017).</li><li>Enjeti, A. K., Lincz, L., Seldon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-maleimide+as+a+generic+stain+for+detection+and+quantitation+of+microparticles.">Bio-maleimide as a generic stain for detection and quantitation of microparticles.</a> Int J Lab Hematol. 30 (3), 196-199 (2008).</li><li>Headland, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-derived+microvesicles+enter+cartilage+and+protect+the+joint+in+inflammatory+arthritis.">Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis.</a> Sci Transl Med. 7 (315), (2015).</li><li>Andersson, T., Dahlgren, C., Lew, P. D., Stendahl, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+expression+of+fMet-Leu-Phe+receptors+on+human+neutrophils.+Correlation+to+changes+in+the+cytosolic+free+Ca2++level+and+action+of+phorbol+myristate+acetate.">Cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Correlation to changes in the cytosolic free Ca2+ level and action of phorbol myristate acetate.</a> J Clin Invest. 79 (4), 1226-1233 (1987).</li></ol>

作者没有任何与此通信相关的利益冲突。

本文介绍了中性粒细胞衍生 MPs 的分离和鉴定的协议。必须考虑到这一程序取得成功的几个关键关键点。首先, PMNs 必须隔离新鲜, 并在2小时内的隔离实验中使用, 以防止自发活化和脱粒。所有的 PMNs 在隔离和刺激点的处理必须在冰上执行, 以防止激活和过早 MP 释放12。第二, 离心为1小时或以上, 最大限度地提高了国会议员的造粒. 第三, 对于流式细胞术的分析, 必须用仪器指定的荧光珠组合正确校准仪器 (珠子类型调整到特定工具) 来正确定义 MP 大小。例如, 当一个流 cytometers 制造商建议使用 FSC 珠子作为与尺寸相关的参数时, 另一些则被优化为 SSC 珠子。此外, 用于 MP 分析的流式细胞仪必须配备升级后的电子设备, 以最佳地解决来自背景噪声的 MPs。
为正确识别 MPs 除了尺寸参数外, 强烈建议使用上述程序中描述的荧光染色。此外, 准备和隔离过程中的所有解决方案都应通过0.1 µm 过滤系统进行过滤, 以最小化尘埃粒子或其他沉淀, 从而在流式细胞仪采集过程中增加背景噪声。重要的是, 应考虑 MPs 的贮存条件。小证据8和我们自己未发表的观察表明, MP 冻结导致膜破裂和 MP 断裂。最后, 不同的中性粒细胞活化条件和时机可能改变, 提高 MP 产量。
此方法有一些限制。从骨髓中分离小鼠 PMNs 通常不纯 (~ 85–90%), 被其他免疫细胞 (主要是单核淋巴细胞) 所污染, 这是由流细胞分析确定的。因此, 由此产生的分离可能包括少量的 MPs 释放的其他免疫细胞。可供选择的, 包括通过商业可用的磁珠隔离 PMNs, 但是, 这将是一个更长的和大大昂贵的程序。最后, 如果没有荧光标记, 除了尺寸参数外, 从尘埃或其他可溶或类似大小的微粒中, 可以污染样品的 MPs 的最终分化是具有挑战性和易出错的。
今后, 对带有特定标记的中性粒细胞进行分类将有助于在特定刺激条件或疾病模型下阐明 MP 的组成, 并希望能够将 MPs 作为诊断标志物和潜在的治疗靶点, 以治疗炎症性疾病。

最近, 来自细胞细胞质或等离子膜的 "微粒/微泡" 已成为极大的科学兴趣, 因为新兴的数据表明, 这些结构, 范围从 50-1, 000 nm 直径, 可以携带生物信息和作为一种非规范化的蜂窝通信方法。免疫细胞衍生的 MPs, 特别是中性粒细胞 (PMNs) 所产生的, 对于 PMNs 在宿主防御中的重要作用是很有兴趣的1,2, 炎症反应3, 伤口愈合4. 迄今为止, 许多报告都显示了中性粒细胞-mps5的亲炎症和抗炎作用, 表明了 mps 的潜在环境、疾病、物种和器官特定作用。
本通信中描述的协议为研究 MPs 在健康和疾病中的作用提供了一种成本效益高、创新性和适应性强的方法。它们适用于许多模型有机体, 器官和刺激条件。他们允许鉴定几种类型的 MPs, 并可用于未来, 以解决他们的亲炎和抗炎功能。例如, 这里描述了如何研究中性粒细胞-MPs 在上皮创面愈合中的作用体外和体内。提出了分离小鼠骨髓源性 PMNs 的协议, 并对先前描述的方法6进行了一些修改。
此外, 本研究中描述的协议可以通过两种互补的方法来检测和表征中性粒细胞 (MPs) 的特异标记物: 西方印迹和流式观察仪。我们发现使用标准协议5的 MPs 的免疫印迹法是容易和可靠的, 然而, 最近的进展, 流式细胞仪的灵敏度和改进的信噪比现在允许进一步分析 MPs 使用这种方法。本研究中所述的协议包括最近的研究成果和建议, 如对离心速度和时间的修改, 添加采样过滤和冻结/存储条件7,8, 以及如何减少 "背景噪声", 提高中性粒细胞的检测极限, 并区分不同大小的 mps。

<table><tbody><tr><td>DMEM</td><td>Corning</td><td>10-041-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (Heat inactivated)</td><td>Atlanta Biologics</td><td>S11150</td><td></td></tr><tr><td>Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td><td>GIBCO</td><td>15140</td><td></td></tr><tr><td>0.5 M EDTA</td><td>Fisher</td><td>BP2482</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888</td><td>Sterile and filtered through a 0.1 micron filter unit</td></tr><tr><td>Sterile filtered Histopaque 1077</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>Sterile filtered Histopaque 1119</td><td>Sigma</td><td>11191</td><td>Density 1.119 g/ml.&amp;nbsp; Solution of polysucrose and&amp;nbsp; sodium diatrizoate. Bring to room temperature</td></tr><tr><td>Phosphate Buffered Saline (PBS) without Calcium and Magnesium</td><td>Cellgro</td><td>210-40-CV</td><td></td></tr><tr><td>Ethyl Alcohol (200 proof)</td><td>Decon labs</td><td>2701</td><td></td></tr><tr><td>HBBS</td><td>Corning</td><td>21-022-CV</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Sigma</td><td>T8154&amp;nbsp;SIGMA</td><td>Diluted 1:2 for cell viability counting</td></tr><tr><td>Isoflurane</td><td>Abbot</td><td>50033</td><td></td></tr><tr><td>Anti-human CD11b-APC conjugated</td><td>Biolegend</td><td>301350</td><td>Clone ICRD44 (Final concetration: 1 &amp;micro;g/mL)</td></tr><tr><td>BODIPY FL</td><td>Invitrogen</td><td>B10250</td><td>N-(2-aminoethyl) maleimide&amp;nbsp;</td></tr><tr><td>Annexin V Binding Buffer</td><td>Biolegend</td><td>422201</td><td></td></tr><tr><td>Calibration Beads for Flow Cytometry</td><td>BioCytex</td><td>7803</td><td></td></tr><tr><td>FITC Annexin V</td><td>Biolegend</td><td>640906</td><td></td></tr><tr><td>Polymorphprep</td><td>Axis-Shield PoC AS</td><td></td><td>Sodium diatrizoate/Dextran 500, density 1.113 g/ml</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Labs</td><td></td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Corning</td><td>430053</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>25 ml serological pipettes</td><td>Celltreat</td><td>229225B</td><td></td></tr><tr><td>10 ml serological pipettes</td><td>Celltreat</td><td>229210B</td><td></td></tr><tr><td>5 ml serological pipettes</td><td>Celltreat</td><td>229205B</td><td></td></tr><tr><td>Pasteur pipettes&amp;nbsp;</td><td>BD Falcon</td><td>357575</td><td></td></tr><tr><td>25 G x 5/8 in. Needles (precision glide needles)</td><td>BD</td><td>305122</td><td></td></tr><tr><td>100 &amp;micro;m cell strainers</td><td>Celltreat</td><td>229485</td><td></td></tr><tr><td>Vacutainer tubes</td><td>BD</td><td>367251</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LSR Fortessa Special Order Research Product (SORP)</td><td>BD</td><td>(SORP)</td><td></td></tr><tr><td>Swinging bucket centrifuge</td><td>ThermoFisher Scientific</td><td>75007210</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>(L8-80 M)</td><td></td></tr><tr><td>Micro-centrifuge</td><td>ThermoFisher Scientific</td><td>&amp;nbsp;VV-17703-15 (Fresco 17)</td><td></td></tr><tr><td>Swinging bucket centrifuge</td><td>ThermoFisher Scientific</td><td>&amp;nbsp;75004503 (Megafuge 40R)</td><td></td></tr><tr><td>Biopsy forceps, 28 cm</td><td>Storz</td><td>27071zj</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image J</td><td>National Institute of Health</td><td>Open source&amp;nbsp;</td><td></td></tr></tbody></table>

isolation and characterization of neutrophil-derived microparticles for functional studies

中性粒细胞衍生微粒-MPs) 是脂双层, 球形微泡大小不等, 直径从 50–1,000 nm。MPs 是细胞对细胞通信和信号机械的一个新兴的、重要的组成部分。因为他们的大小和他们释放的性质, 直到最近 MP 存在被忽略了。然而, 随着技术和分析方法的改进, 它们在健康和疾病中的作用正在出现。这里提出的协议旨在通过流式细胞术和免疫印迹法分离和表征中性粒的 MPs。同时给出了几个实现实例。这些 MP 隔离协议是快速、低成本的, 不需要使用昂贵的套件。此外, 它们允许在隔离后对 MPs 进行标记, 以及在 MP 释放之前对源细胞进行预先标记, 使用膜特异荧光染料进行流式细胞仪的可视化和分析。然而, 这些方法有几个限制, 包括 PMNs 和 MPs 的纯度和复杂的分析仪器的需要。一个高端流式细胞仪是需要可靠地分析 MPs 和尽量减少假阳性读数由于噪声或自动荧光。所述协议可用于分离和定义 MP 合成, 并在不同刺激条件下表征其标记和组成的变化。可利用大小异质性研究膜颗粒与外来体的含量是否不同, 以及它们是否在组织稳态中发挥不同作用。最后, 在对 MPs 的分离和鉴定之后, 可以探讨它们在细胞反应和各种疾病模型 (包括炎症性肠病或急性肺损伤) 中的作用。

从人和鼠标 PMNs 隔离的 MPs 的代表性流细胞分析显示在图 1中。中性粒细胞的大小异质性可以通过与已知大小的珠子的比较来评估, 如图 1A、B中所示. 注意, 小鼠和人的 mps 在大小非均质上没有显著差异。同样, 使用流式细胞术和荧光标记, 可以确定由老鼠或人的孤立的 MPs 所选择的蛋白质/s 的表达。例如, 磷脂丝氨酸 (PhS) 的表达可以通过蛋白 V 染色检查。小鼠骨髓中性粒细胞-MPs 显示在图 1C, D中。一些选择标记的表达也可以评估为蛋白 V 和 CD11b 染色的人 MPs,图 2A, B。另一种检测中性粒细胞的方法是通过流式体术, 在刺激之前染色新鲜隔离的 PMNs, 如图 2C所示。例如, 中性粒细胞染色的脂质标记 n-(2-氨基乙基) maleimide-FITC 之前, fMLF 刺激导致释放的绿色 MPs, 可以很容易地检测到流动。注意, 当染色与 n-(2-氨基乙基) maleimide-FITC 以前被用来标记和检测从外周人类血液中获得的 MPs10或注射到动物11中, 使用其他标记来标记目的体外或体内应用程序应由每个调查人员确定。除了流式细胞术, 中性粒蛋白-MPs 可以通过免疫印迹法来分析感兴趣的蛋白质。如图 3中所示, 从人类 PMNs 中获得的由几个已知的激活剂刺激的 MPs, 被检查为主要炎症 (基质 metallopeptidase 9 (MMP-9) 和髓 (髓过氧化物)) 的表达, 并抗炎 (蛋白 A1) 分子。从代表性的 immunoblots, 中性粒细胞刺激与 IFNγ (50 ng/毫升), 200 nM (1 µM) 导致 MPs 表达不同程度的 MMP-9。然而, 在刺激与 fMLF (5 µM) 之前, 只有肌动蛋白细胞骨架的 Latrunculin B (1 µM) 的摄动, 导致中性粒酶的大量存在。同样, 在描述的活化条件下, 在 MPs 上检测到不同水平的蛋白 A1 (高至无检测)。这些结果表明, 中性粒细胞-MP 组成是刺激依赖。
最后,图 4显示了如何使用孤立的中性粒细胞 MPs 研究创伤愈合体外和在体内结肠损伤。中性粒细胞-MPs 可以添加到擦伤受伤的上皮细胞膜在文化, 在那里愈合可以监测的影像采集在预先确定的时间点。MPs 可以进一步被杏仁直接进入结肠伤口, 这是由活检钳和内窥镜成像9产生的, 并可评估其对愈合的影响。对于体外和体内分析伤口愈合, 被造成的伤口图像立即获得后受伤 (或其他指定), 并持续通过愈合过程在预先确定的时间点。利用商用图像分析软件, 测量伤口面积 (尺寸) 的变化, 并用于确定创面闭合率。中性粒细胞的应用, 其中含有过氧化物酶的培养上皮膜或体内结肠伤口有不利的影响, 导致延迟愈合9。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56949/56949fig1.jpg"> 图 1.流式细胞仪分析中性粒径和表面标记物.(A) 流式细胞仪优化控制珠 (参见材料表) 已知大小和散射值显示在 SSC 和 FSC 正交表示法中。该珠子用于获得与 b 中所示的中性粒细胞 MP 样品的相对大小比较. (B) 人中性粒体-MPs 被分离和分析用流式术后使用的条件描述的珠子。可以看到 MP 大小的异质性。(C-D)采用流式细胞仪对 fMLF 刺激小鼠骨髓 PMNs 的 MPs 进行了分析。代表性流程图显示无瑕 (C) 或蛋白 v FITC 染色 (D) mps. 矩形区域显示蛋白 V 阳性 mps (FITC 阳性 mps)。侧向散射;前向散射;小灵通: 磷脂丝氨酸。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56949/56949fig1large.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/56949/56949fig2.jpg"> 图 2.流式细胞仪检测中性粒-MP 染色及分析.(A) 无瑕和 (B) 蛋白 V-FITC-和 hCD11b-APC-stained mps. 正方形区域包含一个蛋白 V/CD11b 双阳性的中性粒细胞-mps. (C) 新鲜隔离的人类 PMNs (1 x 106) 被荧光染料染色, n-(2-氨基乙基) maleimide-FITC 和 fMLF 刺激。采用流式细胞仪对 MPs 进行细胞上清液分离。矩形区域包围一个 n-(2-氨基乙基) maleimide FITC 阳性 MP 填充 (M-FITC, Y 轴标签)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56949/56949fig2large.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/56949/56949fig3.jpg"> 图 3.中性粒细胞的组成-MPs 是刺激依赖.(A) 使用 IFNγ、TNFα、fMLF、PMNs 或 latranculin B 的组合 (fMLF LtB) 刺激人的。在 1% SDS 缓冲液中制备了离心和蛋白裂解物, 分离出细胞上清液。蛋白质是由大小电泳在10% 聚丙烯酰胺凝胶, 转移到硝化纤维膜, 并探讨了 MMP-9, 髓过氧化物酶, 或蛋白 A1 原抗体, 其次是适当的酶共轭继发抗体。(B) 人中性粒细胞的代表性电子显微图像-MPs 描述大小异质性。外切 < 100 毫微米在大小由白色箭头指示。刻度条 = 250 毫微米 (左和右面板)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56949/56949fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56949/56949fig4.jpg"> 图 4:使用孤立的中性粒细胞-MPs 在研究 PMNs 在上皮伤口愈合中的作用.(A) Caco-2 BBe 人肠道上皮细胞被镀到融合、擦伤伤, 并受来自 300万 PMNs 的 MPs 的伤害, 这一情况在受伤后立即增加。有代表性的图像显示, 在控制 (左面板) 和中性粒细胞 MP 治疗的上皮干细胞 (右面板) 伤口闭合 (48 小时后受伤)。刻度条 = 100 µm. (B) 检查中性粒细胞-mps 对结肠创面愈合的影响体内, 孤立的中性粒细胞-mps 被直接注射到伤口区, 使用内窥镜下的显微注射系统 (24 小时后伤后, 结肠伤口是由虚线和注射针站点概述由一个白色箭头显示)。3天后 (伤后4天) 经内镜成像评估创面闭合。刻度条 = 300 µm. (C) 4 天后, 伤后小鼠被安乐死, 并用剪刀提取结肠黏膜创面, 嵌入最佳切削温度 (o.c. T) 化合物, 冷冻液氮。八微米切片的伤口被染色的 e-钙黏蛋白 (绿色) 和核染色 DAPI (蓝色), 以评估重新上皮 (上部板) 的水平, 或 DAPI (蓝色) 和 Ki67 (红色), 以可视化的总和增殖上皮细胞在伤口边缘 (下部板)。缩放条 = 50 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56949/56949fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies at JoVE.com

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

本文介绍了新的协议, 以分离和表征从人和小鼠中性粒细胞衍生的微粒。这些协议利用离心、流式细胞术和免疫印迹法技术对微粒含量进行分析, 可用于研究不同细胞类型的微粒在细胞功能中的作用。

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

isolation-characterization-neutrophil-derived-microparticles-for

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  Northwestern University. 本文介绍了新的协议, 以分离和表征从人和小鼠中性粒细胞衍生的微粒。这些协议利用离心、流式细胞术和免疫印迹法技术对微粒含量进行分析, 可用于研究不同细胞类型的微粒在细胞功能中的作用。

视频: Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

科研

生物学

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

生物工程

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

免疫与感染

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 ...

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 ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

用于筛选不同信号通路的中性粒细胞功能检测

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

作者聚焦:使用 NeutroFun 筛选方案平衡中性粒细胞功能评估的速度、成本和准确性 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

一套快速了解中性粒细胞功能的筛查技术

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

中性粒细胞衍生微粒的分离与鉴定功能研究

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此视频中的章节

嗜中性粒细胞的分离协议

微流体平台从未经加工的全血测定中性粒细胞趋

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

隔离和中性粒细胞的表征与抗肿瘤性质

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

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术