作者感谢伊迪丝·苏斯 - 托比博士为她与激光共聚焦显微镜研究宝贵的帮助。这项研究是由移民吸收部和委员会规划和高等教育委员会的预算的KAMEA程序（LD和AP）的框架下支持。我们也非常感谢来自戴维斯夫人基金会博士后研究奖学金的研究员（OR）的支持。

该协议是根据赫尔辛基宣言批准由当地人权委员会及所有参与者签署了知情同意书。 1.中性粒细胞分离和培养Gφ发展注:应使用无菌组织级脂多糖（LPS） - 免费解决方案，在生物安全层流罩内进行的所有步骤。不添加抗生素，细胞因子或生长因子的罗斯威尔公园纪念研究所（RPMI）-1640培养基。 <ol><li>使用无菌头皮静脉集年轻健康的成年人在获得至少40毫升的静脉血。抽血到含有乙二胺四乙酸K 3盐真空采血管（K 3 EDTA），轻轻混匀。保持血液在室温下。 </li><li>隔离使用聚蔗糖两步连续密度梯度中性粒细胞在1.119和1.077克/毫升。使用前使至室温的解决方案。 注意:在离心分离，红血细胞（红细胞）由多聚蔗糖和沉积物迅速聚集。的单核细胞（单核细胞/淋巴细胞）在上部等离子体之间发现/多聚蔗糖-1,077接口，而嗜中性粒细胞中发现只是红细胞之上，在聚蔗糖-1,077 / 1119接口（ 见图2）。该方法允许来自同一个体的单核细胞和嗜中性粒细胞的同时分离。 </li></ol><img alt="图2" src="/files/ftp_upload/54826/54826fig2.jpg" /> 图2:从全血中性粒细胞隔离。多聚蔗糖在1.077克/ ml的小心地层叠在聚蔗糖 - 1.119克/ ml的顶部，以形成一个不连续梯度。然后将稀释的全血层叠在聚蔗糖 - 1.077的顶部。将试管立即经受在700 xg离心离心分离30分钟，在室温下无制动。三个不同的频带指出。 （A）的单核细胞，（B）的多形核细胞（PMN），和（C）的红血细胞（RBC）在管的底部。 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol style="margin-left: 40px;"><li>添加12毫升聚蔗糖-1119到50ml无菌聚丙烯离心管的底部。 </li><li>仔细层12毫升聚蔗糖-1077到聚蔗糖-1119。 </li><li>稀释10 - 19 ml完整血的24毫升血用离子自由磷酸盐缓冲盐水含有2％热灭活的胎牛血清（HI-FCS）的最终体积（PBS）中。仔细层24毫升稀释的全血的上管的上部梯度。 </li><li>离心机在700×g离心，在室温下30分钟 - 无制动器（20 24℃）。 注意:离心在较低温度下可能会导致细胞聚集和恢复差。 </li><li>小心地从离心分离机取出管withouŧ干扰梯度。两个遮光层应观察（A:单核细胞和B:PMN，在图2中描绘）。 </li><li>吸并丢弃这层流体达0.5厘米以上层A.传输（或丢弃）细胞管标有"单核"。 </li><li>吸并丢弃剩下的流体可达0.5厘米以上层B.从该层转移细胞到标有"PMN"一管。 </li><li>从每两个梯度管池PMN并用含有2％的HI-FCS至30毫升的最终体积的PBS洗涤。离心12分钟，在200 XG，弃上清并丢弃。 </li><li>摆脱污染红细胞（RBC），加3毫升低渗0.2％冰冷的无菌氯化钠，同时通过用1毫升无菌吸管尖轻轻拉伸进出重悬浮沉淀。保持在冰上30秒。 </li><li>后30秒，通过加入3毫升无菌1.6％冰冷的NaCl到管恢复等渗。 </li><li>到6毫升等渗盐水，加入6米预热（37℃）l的RPMI-1640培养基补充有2％的HI-FCS和离心机在250×g离心12分钟。弃去上清液。该PMN颗粒应清洁RBC污染。 注:如果RBC污染时，PMN颗粒出现微红。 </li><li>如果某些污染RBC仍然存在，重复步骤9和10多次。 </li><li>悬浮在4ml RPMI-1640添加有10％的HI-FCS细胞沉淀并计数细胞用台盼蓝排除，以确定其浓度和存活率。 </li><li>浓度调整至1.25 - 1.5×10 6 PMN / ml的（取决于实验需要），和板1.0毫升/孔在24孔板中。 注:在人口粒细胞中性粒细胞的纯度总是超过95％，在五月的Grunewald，姬姆萨染色和光镜的评估。 </li><li>播种后，放置在一个单元 湿润的5％CO 2培养箱在37℃。 </li><li>轻轻吸的一半更换介质每3天培养基并加入新鲜RPMI-1640培养基的相同体积补充有10％的HI-FCS中。使用脂多糖自由溶液和化合物和低内毒素水平的HI-FCS（0.05毫微克/毫升或更小）。 注:温和介质变化是势在必行自Gφ，这在培养开发不牢固地附着于培养皿和剧烈洗涤也可以洗掉的显影单元。 PMN培养后第4天，这取决于血液供体 - Gφ的外观是在3明显。在培养7天后，当Gφ在尺寸上非常大的 - 大多数这里描述的分析和测定都6之间执行。应当注意的是，除了1 - 10纳克/ ml的LPS的RPMI-1640培养基没有影响Gφ发展中培养。 14 </li></ol> 2.激光共聚焦显微镜<ol style="margin-left: 40px;"><li>从新鲜分离的嗜中性粒细胞制备细胞离心涂片16，并从7天开发Gφ培养（第1节准备）。 注:为了增加菜的各种分析Gφ的浓度，轻轻地取下媒体的一半。确保Gφ中未除去培养基通过在光学显微镜下检查介质进行检测。然后，集中吸管中剩余删除轻轻地粘附Gφ。离心介质为在200 xg离心10分钟，并且重悬沉淀在100 - 120微升培养基。 <ol><li>使用100 - 120微升包含每张幻灯片细胞的培养基中。从各处理制备重复或三次重复幻灯片。旋转在84×g的7分钟。 </li></ol></li><li>干纺出细胞，并固定，用4％多聚甲醛将细胞在室温下化学罩10分钟。洗3次，用PBS（约100微升每洗涤几秒钟）。对于细胞内染色，透化，用0.5％的Triton X-100的PBS细胞在室温下10分钟，并用PBS洗涤5次。 注:在所有阶段，使用适当的缓冲/解决方案VOLU我以覆盖载玻片上的细胞的周边。使用确定细胞外围疏水屏障笔。 注意:多聚甲醛是有毒的。避免与皮肤和眼睛接触。穿戴合适的个人防护装备。 </li><li>块细胞在RPMI-1640培养基10％正常山羊血清，并在4ºC或在室温下进行40分钟孵育过夜。用PBS洗涤。 </li><li> 100稀释（〜100微升）:用单一抗体（AB）或在1小鼠和兔的初级抗体（ABS）的组合孵育。在4°C - （20小时18）孵育过夜。 注意:在此，包含小鼠单克隆ABS:抗-CD14，抗CD63，抗CD66b，抗CD1C，抗CD15和抗细胞色素b-245轻链（p22- PHOX识别）。兔多克隆绝对包括:抗CD68，抗CD36，抗LC3B，抗髓过氧化物酶，抗中性粒细胞弹性蛋白酶（NE），以及抗NOX2 / gp91- PHOX腹肌。同种型对照包括纯化的小鼠IgG1和IgG2的，和兔IgG。 PreparÈ根据制造商的说明在ABS和使用适当的体积（约100μl），以覆盖所述电池的周边。 </li><li>洗涤细胞并用1/400二抗的Cy2-CF（488A）缀合的山羊抗兔IgG（绿色）孵育/或Cy5的（CF 647）缀合的山羊抗小鼠IgG（红色），在室温下搅拌40分钟。 注:根据制造商的说明稀释，并准备腹肌。 </li><li>在洗涤后，装入滑动带安装介质，含有4&#39;，对核染色6-二脒基-2-苯基吲哚（DAPI）中的一个降低，然后立即放置盖玻片。 </li><li>通过使用荧光显微镜和计划的Apo 40X浸油物镜共焦激光扫描系统进行分析的幻灯片。制备的幻灯片之后执行于30分钟至2小时的分析或在4℃下保持过夜。 <ol><li>计算单元面积和使用成像软件（ 例如，图像J）的荧光强度。对于共定位，QUAntify使用曼德斯重叠系数（MOC）17软件。 注:只有具有MOC细胞&gt; 0.6可以被认为是与显著共定位的细胞。 </li></ol></li></ol> 3.中性粒细胞的轮回跨内皮细胞:巨噬细胞IL-8的影响（Gφ）形成注:使用24孔渗透性细胞培养插入的电池轮回检测。 <ol style="margin-left: 40px;"><li>涂层用150微升纤连蛋白的插入物以50微克/ ml的浓度的上腔室，并保持在室温下30分钟。 </li><li>添加到上腔室5×10 4将EA.hy926内皮细胞/孔，再悬浮于150微升配制Dulbecco氏改良的Eagle氏培养基（完全培养基）的。 注:确保内皮细胞单层汇合后使用。 </li><li>到下腔室中，添加完全培养基的700微升。 </li><li>将渗透细胞培养在簇托盘和培养将EA.hy926内皮细胞2天插入于37℃在5％CO 2。 注:在并行，第二天准备新鲜的中性粒细胞（如第1节）。 </li><li> 2天后，在该插入物的下部和上部腔室更换介质。 <ol><li>到下室中，添加补充有10％III-FCS和白细胞介素（IL）-8 RPMI-1640培养基在50纳米/ ml的终浓度。不要添加IL-8的控制下腔。 </li><li>到每个上部室，在100μlRPMI-1640培养基的补充有10％H-FCS添加10 6新鲜的PMN。 </li></ol></li><li>孵育群集盘在37℃下在5％CO 2 90分钟。 </li><li> 90分钟孵化后，从上层的细胞和下腔分开并计数各亚群。在每个腔室的总细胞的百分比明示细胞加入。 注:小心地从上chamb除去细胞器通过以避免去除内皮细胞和转移到无菌管中轻轻吹打。通过移液并用500μl和转让下部腔洗涤到第二无菌管中删除从下室中的细胞。 </li><li>如在步骤中指定池10 6个细胞从transmigrating（下室）和非迁移（上室）PMN馏分并培养每7天无生长因子的几个孔14 - 16（第1部分）。 </li><li>旋转细胞在载玻片上16和第2条所描述的分析通过共聚焦显微镜各培养条件发达的细胞。 </li></ol>

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作者什么都没有透露。

巨噬细胞（Gφ）是新定义的表达基本和比嗜中性标记物诸如CD66b / CD15 / CD63 / MPO / NE嗜中性粒细胞衍生的细胞的亚群。这种类型的嗜中性粒细胞衍生的吞噬细胞的未在文献中之前所描述。不像是短暂的，凋亡中性粒细胞，Gφ是膜联蛋白-V-阴性和显示扩展寿命。然而，象嗜中性粒细胞，Gφ也内在颗粒，并且响应于这些颗粒和对PMA产生NADPH氧化酶 - 依赖性ROS。然而，他们的能力内化脂蛋白，从而产生活性氧是Gφ的独特功能。 14 被证明有许多因素影响其发展的文化。在生长培养基中缺乏外部的细胞因子或生长因子是必不可少的（具体的GM-CSF / IL-4）。然而，中性粒细胞对IL-8的迁移证明那些dev目录之间的区别因素私奔到Gφ和那些没有。另外，从细胞凋亡的中性粒细胞产生的碎片的内化，自噬蛋白（LC3B）和功能NADPH氧化酶的表达，都证明是当务之急的发展，因为它们的抑制防止Gφ形成（ 图1）。显然，从嗜中性粒细胞产生的这些巨细胞发育的不同之处的单核细胞/巨噬细胞谱系是表征巨细胞形成。与不同的慢性炎性疾病，20，21相关的一种形式的多核巨细胞而嗜中性Gφ描述此处通过自噬发展，由吞噬细胞残渣和保持大部分单核在其整个发展，14（很少然而，有时一第二核可以观察到）。此外，多个控件建立了嗜中性粒细胞的起源:（1）exprePMN具体neutropilic标记的裂变和不存在的树突和单核细胞谱系标记物，（2）它们在单核细胞阻碍发展/共培养，（3）从巨噬细胞（如由活细胞成像和时间其在培养运动的不同图案-lapse显微镜），14（4）他们的光坚持塑料餐具和（5）从单纯的CD15 + / CD14的发展- PMN通过流式细胞仪获得的。 一些体外识别功能可以给我们的线索，它们在体内的潜在功能。例如，Gφ的能力消耗大量嗜中性粒细胞颗粒和碎片，大液泡的存在，并表达LC3B -这有助于通过监管的互动与先天免疫信号通路消炎的自噬蛋白质，22 -所有这些都支持清除能力。因此这些发现还表明，Gφ可能在炎症部位，其中所述巨噬细胞/直流系统不足或淹没进行运作，从而有助于炎症的分辨率。这个概念可能的事实，在混合单核细胞/嗜中性粒细胞培养物Gφ发展受到阻碍的支持。 14此外，鉴于Gφ表达氧化低密度脂蛋白清道夫受体（CD36，CD68），内化脂蛋白，并响应于它产生活性氧，可以指示他们参与动脉粥样硬化的进程来解决发炎。因为Gφ只从其中迁移的朝向的IL-8的中性粒细胞和嗜中性粒&#39;跨内皮单层向IL-8的表示中性粒细胞募集到急性炎症部位轮回开发的，这一发现还可以支持抗炎功能。相反，Gφ在某些炎症性疾病的表现可能使他们能够履行颗粒成分和活性氧，从而促使每sistent炎症和组织损伤。 20但是，总体而言，他们的自噬能力表示Gφ有可能参与减少炎症反应，而不是延续它。 有趣的是，我们最近发现Gφ在人类粥样硬化斑块的存在。 （在准备）。然而，问题大量尚待揭开。例如，是Gφ亲或抗炎？什么是决定在体外或体内的形成和功能的因素是什么？哪些具体的中性粒细胞亚群是促进其发展成Gφ其前体细胞？他们是否与某些病理和相关？总的来说，冒充有趣的问题是他们的原籍和潜在功能。 该协议内。然而关键步骤和缺陷，应牢记。在Gφ我的发展的关键一步小号在培养的细胞因子，生长因子或抗生素的媒体缺乏纯粹的中性粒细胞。另一个关键步骤是排除Gφ污染的单核细胞培养，并确定Gφ的中性粒细胞的起源。因此，通过连续梯度血液分离后，嗜中性粒细胞进一步进行纯化的额外步骤通过流式细胞术使用粒细胞门控和CD15 + / CD14 -的标记。发达Gφ从通过流式细胞术分离进一步纯化没有从那些不进行纯化的该步骤不同的中性粒细胞获得。因此，大多数实验是未经术纯化的步骤的流程由于额外的细胞损失进行。值得注意的是，在某些罕见的情况下一些嗜酸性粒细胞培养中指出。它们的规模基本保持在整个培养周期不变。我们还应该注意到，虽然有许多方法从人血嗜中性粒细胞的分离，这里所描述的方法是我们所用的唯一方法，因此我们不能为嗜中性粒细胞的分离其他可用的方法比较Gφ发展。 在调查Gφ一个主要缺陷从导致无法获得适合各种生化分析纯Gφ人口的足够数量。它是在我们的实验进行的条件基本上是不可能的。首先，Gφ的产率是低的。从1.0×10 6 PMN接种大约100 - 200Gφ七天后发展培养，这取决于血液供体。其次，它是目前的发达Gφ中培养从在盘的其余的中性粒细胞碎片中分离基本上很难。这些限制使得实际上不可能通过生物化学或分子生物学方法分析细胞。因此，该协议的重点是在描述由利用光Gφ识别和功能和共焦显微镜。中性粒细胞在培养自己的形态转变成Gφ还跟着活细胞成像和时间推移显微镜。 14显然，可能需要以实现生物化学或分子生物学方法，克服获得的低产量以及从嗜中性粒细胞"在盘碎片分离可行Gφ大得多血液容量。 总之，我们最近首次描述Gφ的发展中培养，中性粒细胞来源的长寿命吞噬细胞的一个亚群。因此，这是目前可用的，以获得Gφ在培养的唯一方法，虽然上述的两个主要的限制，应克服（在培养获得的Gφ的低产以及无法显影Gφ从嗜中性粒细胞的碎片在该培养基中分离碟）。不过，它们的制备和鉴定，在该协议提出，为ESSENTIAl对于感兴趣的炎症反应和中性粒细胞生物学和可塑性，以进一步调查Gφ的潜在意义和作用的科学家。

中性粒细胞（PMN）构成了血液内白血球的最多的人口，作为抵御通过产生多种细胞毒性分子侵入的病原体的第一道防线。传统的观点早就被血液循环，短暂的，专业的吞噬细胞，这是第一个到达的急性炎症部位，以打击感染和援助的病原体和有害微粒的间隙。 1在它们的非激活状态，中性粒细胞是组成性致力于凋亡。当从血液到炎症部位迁移，中性粒细胞活化进行化解炎症。它们吞噬和杀死侵入的微生物，通过产生细胞毒性分子作为活性氧（ROS），溶解酶，如嗜中性弹性蛋白酶（NE）和具有强效的杀微生物活性的组织蛋白酶的阵列。为了陷阱病原体，中性粒细胞也释放外陷阱（英语教师），它是由含有抗菌肽和各种裂解酶核染色质的线程。然而，中性粒细胞这些细胞毒性分子的释放失控还可能延续炎症反应并诱发周围组织损伤。 2因此，由巨噬细胞的凋亡嗜中性粒细胞的一种有效的清除率（Mφ）和树突细胞（DC）是至关重要的，以解决炎症。 3，4，5，6 然而，在最近几年，它已成为越来越明显的是，中性粒细胞是高度通用的细胞，其功能远远超出吞噬病原体和杀戮。 6，7在经历底漆或活化，中性粒细胞的可塑性也逐渐受到关注。例如，细菌和分枝杆菌攻击嗜中性粒细胞被证明分泌白细胞介素（IL）-10和控制炎症反应，这表明的免疫调节应答的存在。 8有丝分裂后嗜中性粒细胞显示出反分化为巨噬细胞样细胞，或DC样细胞消化和当与细胞因子和生长因子，9，10这样处理呈递抗原片段，在整合先天和适应性服务关键作用响应。 3，6激活由生长因子促进凋亡中性粒细胞或细胞碎片，从而的吞噬，促进发炎部位的杂物清除和炎症消退，3,9特别是在巨噬细胞/ DC通关系统不足或不堪重负，11，12这表明潜在的&#39;自律&#39;，以帮助重建解决炎症反应。这一点，因为细胞凋亡是稳压自死亡能够抑制细胞毒性化合物的细胞外释放，从而防止伤害周围组织的一种形式。 6 生存时间延长是嗜中性粒细胞活化的另一个特征和通过用各种宿主衍生的因子如粒细胞集落刺激因子（G-CSF），粒细胞 - 巨噬细胞集落刺激因子（GM-CSF），炎性细胞因子，如干扰素（治疗证明IFN）-γ，肿瘤坏死因子（TNF）-α和/或病原体衍生的产品，从而，使嗜中性粒细胞，以调节其生存反应。 6事实上，嗜中性粒细胞存活是其塑性的先决条件，并用其来执行吞噬能力相关联。 6,13因此，也显示出与依赖新生表型和功能的改变相关联DED上通过诱导参与嗜中性粒细胞寿命延长新蛋白的合成上调的基因表达，和降低凋亡。 10 不同于中性粒这是短暂的，组成在培养进行细胞凋亡，或细胞因子/生长因子激活嗜中性粒细胞，如上所述，已延长寿命，我们最近发现嗜中性粒细胞的一种新的，小亚群，在延长的标准培养自发发展从新鲜分离的人血嗜中性粒细胞，而不外部添加细胞因子或生长因子的条件。 14这些嗜中性粒细胞衍生的细胞，其未在文献中之前描述的方法被称为巨噬细胞（Gφ）。该Gφ已扩大文化寿命，它们完全开发的5-7天内，并通过独特的形态特征，表型表达和功能的特点。他们被大大放大，由于autophago中性粒细胞死亡残存，空泡化的吞，并包含吞噬溶酶体。该Gφ表达了中性粒细胞特异性标志物颗粒-分化（CD）66B的集群中，嗜天青颗粒标记- CD63和髓过氧化物酶（MPO）和额外的嗜中性粒细胞标志物如CD11b的，NE，CD15，NADPH氧化酶亚基gp91- PHOX和p22- PHOX和自噬标记-LC3BII。 14，15在功能上，它们积极地卷取乳胶珠和酵母聚糖颗粒，以及响应于酵母多糖和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激产生的ROS。有趣的是，不像新鲜的嗜中性粒细胞，Gφ也集中表达了清道夫受体CD68和CD36，卷取氧化低密度脂蛋白（氧化低密度脂蛋白），并响应与氧化低密度脂蛋白的刺激产生活性氧。此外，Gφ是泯灭单核细胞系标记CD14，CD16和CD163或树突状标记CD1C和CD141的。此外，PHAgocytosis和自噬和可能的功能NADPH氧化酶是其发展的先决条件。这因为，吞噬抑制剂cytochalsin B，自噬抑制剂3 - 甲基腺嘌呤（3-MA）和巴弗洛霉素（BafA1）和NADPH氧化酶抑制剂 - 二亚苯基碘（DPI） - 阻碍其发展。此外，单核细胞/中性粒细胞共培养，以及暴露于阻碍了他们的发展间歇性缺氧，而中性粒细胞适应缺氧的持续明显。 14,15他们的文化发展建议的图1 .The协议说明在本文介绍一步一步Gφ从制剂新鲜分离的循环人体血液中中性粒细胞，它们的发展，鉴定和一些基本特征。该协议可以用于进一步调查和揭示广谱的作用，这些新描述和有趣的嗜中性粒细胞衍生的，以便Gφ到characterizË它们的意义和潜在功能。 <img alt="图1" src="/files/ftp_upload/54826/54826fig1.jpg" /> 图1: 在7天中性粒细胞培养巨细胞发展的示意图。有人建议，在炎症部位（1）的中性粒细胞经历凋亡的细胞死亡，和（2）释放的膜包围含有核碎片，颗粒剂（绿色和红色的点），该触发自噬机制片段，以及其它亚细胞组分。在长期嗜中性粒细胞培养物缺乏细胞因子或生长因子由内在凋亡小体和嗜中性粒细胞碎片（3）巨噬细胞（Gφ）发展，同时维持功能性的NADPH oxidase.They的特征在于各种嗜中性CD66b + / CD63 + / MPO + / CD15 + / CD11b的+ / NE标记，大吞噬体包封的颗粒和细胞碎片，清道夫受体CD36和CD68。 GB1;大多是单核细胞，能够也内在各种粒子和氧化的LDL和产生活性氧。液泡填充Gφ的膜包含LC3B（标记为深蓝色），autophagosomal膜的标记物，这表明自噬和巨噬细胞的形成之间的严格关系。 Gφ不要在包含GM-CSF / IL-4的培养基培养。此外，抑制剂如NADPH氧化酶抑制剂 - 二亚苯基碘鎓（DPI），该自噬抑制剂3-甲基腺嘌呤（3-MA）和巴弗洛霉素（BafA1）和吞噬抑制剂细胞松弛素B（的Cyto B）中废除其形成。 （4） 在体内潜在Gφ功能可能包括抗或促炎症性和动脉粥样硬化进程的参与（这个数字是基于我们的研究结果14日 ，15日和伴随的社论B的修改erton 20）。 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Sterile scalp vein set (21GX3/4)&amp;nbsp;</td><td>Bio Diagnostics Ltd.</td><td># 20080312</td><td>A strile needle for venipancture</td></tr><tr><td>VACUETTE HOLDEX Single-Use Holder PP</td><td>Greiner Bio-One</td><td># 450263</td><td>For securing during venipuncture&amp;nbsp;</td></tr><tr><td>VACUETTE Tube K3E K3EDTA (16 x 100/9 mL)</td><td>Greiner Bio-One</td><td># 455036</td><td>Sterile tube for blood collection</td></tr><tr><td>Nunclon MultiDish (24 well x 1 ml)</td><td>Thermo Scientific</td><td># 142475</td><td></td></tr><tr><td>Polypropylene conical centrifuge tube (50 ml)</td><td>Greiner Bio-One</td><td># E14103PJ</td><td></td></tr><tr><td>Transwell-24 well (transmigration assay)</td><td>Corning</td><td># CA-3415</td><td>Polycarbonate membrane, 6.5 mm diameter, 3 &amp;mu;m pores)&amp;nbsp;</td></tr><tr><td>RPMI-1640 medium</td><td>BioIndustries</td><td># 01-100-1A</td><td>Do not add antibiotics&amp;nbsp;&amp;nbsp;</td></tr><tr><td>EA.hy926 (ATCC CRL2922)&amp;nbsp;&amp;nbsp;</td><td>BioIndustries</td><td># CRL-2922</td><td></td></tr><tr><td>ATCC-formulated Dulbecco&amp;rsquo;s&amp;nbsp; Modified Eagle&amp;rsquo;s Medium</td><td>BioIndustries</td><td># 302002</td><td>complete growth medium</td></tr><tr><td>Polysucrose - Histopaque1119</td><td>Sigma-Aldrich</td><td># 1119-1</td><td>Tissue culture grade</td></tr><tr><td>Polysucrose - Histopaque1077</td><td>Sigma-Aldrich</td><td># 1077-1</td><td>Tissue culture grade&amp;nbsp;</td></tr><tr><td>Phosphate buffered saline (PBS) - ion free</td><td>BioIndustries</td><td># 02-023-1A</td><td>Cell biology and molecular biology grade</td></tr><tr><td>Heat inactivated Fetal calf serum (HI-FCS)</td><td>BioIndustries</td><td># 04-121-1B</td><td>Cell biology grade or tissue culture grade, low LPS</td></tr><tr><td>NaCl</td><td>Sigma</td><td># S3014</td><td>Molecular biology grade, suitable for cell culture</td></tr><tr><td>Paraformaldehyde, 16%</td><td>Electron Microscopy Sciences</td><td># 15710</td><td>Cell bology grade or tissue culture grade (only 16% PFA)</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td># 9002-93-1</td><td>Molecular biology grade&amp;nbsp;</td></tr><tr><td>Normal Goat Serum</td><td>BioIndustries</td><td># 04-009-1</td><td>Cell biology and molecular biology grade</td></tr><tr><td>Trypan blue</td><td>BioIndustries</td><td># 031021B</td><td>Tissue culture grade&amp;nbsp;</td></tr><tr><td>May-Gr&amp;uuml;nwald</td><td>Sigma-Aldrich</td><td># MG500</td><td>Cell biology grade-(procedure No GS-10)</td></tr><tr><td>Giemsa stain Kit</td><td>Sigma</td><td># 48900</td><td>Cell biololgy grade-(procedure No GS-10)</td></tr><tr><td>Fibronectin</td><td>BioIndustries</td><td># 03090105</td><td></td></tr><tr><td>Human Interleukin-8 (CXCL8)</td><td>PeproTech</td><td># 200-08-5</td><td></td></tr><tr><td>Anti-CD14 (clone 5A3B11B5)</td><td>Santa Cruz Biotechnologies</td><td># sc-58951</td><td>Mouse IgG2b; expressed by&amp;nbsp; monocytes</td></tr><tr><td>Anti-CD63 (clone MX-49.129.5)</td><td>Santa Cruz Biotechnologies</td><td># sc-5275</td><td>Mouse IgG1; expressed by neutrophils</td></tr><tr><td>Anti-CD66b (clone 80H3)</td><td>AbD Serotec</td><td># MCA216</td><td>Mouse IgG1; expressed by neutrophils</td></tr><tr><td>Anti-CD1c (BDCA-1) (clone AD5-8E7)</td><td>MACS Miltenyi Biotec</td><td># 130-090-695</td><td>Mouse IgG2a; expressed by&amp;nbsp; dendritic cells</td></tr><tr><td>Anti-CD15 (clone MY-1)</td><td>Abcam</td><td># ab754</td><td>Mouse IgM; expressed by neutrophils</td></tr><tr><td>Anti-Cytochrome b-245 Light Chain (p22-phox) (clone 44.1)</td><td>BioLegend</td><td># 650001</td><td>Mouse IgG2a; to recognize neutrophil NADPH oxidase complex</td></tr><tr><td>Anti-CD68</td><td>Protein Tech</td><td># 16192-1-AP</td><td>Rabbit IgG; to recognize oxLDL scavenger receptor&amp;nbsp;</td></tr><tr><td>Anti-LC3B</td><td>Sigma</td><td># L7543</td><td>Rabbit IgG&amp;nbsp;</td></tr><tr><td>Anti-Myeloperoxidase</td><td>Abcam</td><td># ab45977</td><td>Rabbit IgG</td></tr><tr><td>Anti-Neutrophil elastase</td><td>Calbiochem</td><td># 481001</td><td>Rabbit IgG&amp;nbsp;</td></tr><tr><td>Anti-NOX2 (gp91-phox)</td><td>Abcam</td><td># ab131083</td><td>Rabbit IgG</td></tr><tr><td>Anti-CD36 (SR-B3)</td><td>Novus Biologicals</td><td># NB400-144</td><td>Rabbit IgG</td></tr><tr><td>Purified Mouse IgG1, &amp;kappa; Isotype Control (clone MG1-45)</td><td>BioLegend</td><td># 401401</td><td>Antibody used as isotype control</td></tr><tr><td>Purified Mouse IgG2a, &amp;kappa; Isotype Control (clone MOPC-173)</td><td>BioLegend</td><td># 400263</td><td>Antibody used as isotype control</td></tr><tr><td>Normal rabbit IgG</td><td>Santa Cruz Biotechnologies</td><td># sc-2027</td><td>Antibody used as isotype control</td></tr><tr><td>CF488A Goat Anti-Rabbit IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20012</td><td>Anti-Rabbit IgG with the green fluorescent dye CF488A</td></tr><tr><td>CF647 Goat Anti-Rabbit IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20043</td><td>Anti-Rabbit IgG with the red fluorescent dye CF647</td></tr><tr><td>CF488A Goat Anti-Mouse IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20010</td><td>Anti-Mouse IgG with the green fluorescent dye CF488A</td></tr><tr><td>CF647 Goat Anti-Mouse IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20040</td><td>Anti-Mouse IgG with the red fluorescent dye CF647</td></tr><tr><td>Fluorescent Mounting Medium with DAPI</td><td>Vectashield H-1000; Vector Lab Inc.</td><td># E19-18</td><td>Nuclear staining</td></tr><tr><td>Confocal laser scanning microscope (LSM 700)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Carl Zeiss</td><td>Ser.# 3523000380</td><td>Plan Apo 40X immersion oil objective&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Zeiss CLSM software (ZEN 2010)</td><td>Carl Zeiss MicroImaging GmbH</td><td>version 6.0</td><td>For colocalization analysis</td></tr><tr><td>ImageJ software</td><td>Wayne Rasband, NIH, USA</td><td>version 1.49k</td><td>For determination of cell&amp;nbsp; areas and fluorescence intensity</td></tr><tr><td>Light microscope (Axiovert 25)</td><td>Carl Zeiss</td><td>Ser.# 201060153</td><td>Examination of cells in culture</td></tr><tr><td>Centrifuge (Megafuge 1.0 R)</td><td>Heraeus Instruments</td><td># D-37520</td><td>Cells separation from blood; cytospins preparation</td></tr><tr><td>Inverted fluorescent microscope (Zeiss Axio Observer Z.1)</td><td>Carl Zeiss</td><td>Ser.# 3834001470</td><td>Demonstration of giant phagocytes development</td></tr><tr><td>Temperature-controlled incubation system (Cube&amp;amp;Box)</td><td>Life Imaging Services</td><td></td><td>Temperature control system for microscopes</td></tr><tr><td>High&amp;nbsp;resolution digital CCD camera (AxioCam HRm)</td><td>Carl Zeiss</td><td>Ser.# 117090279</td><td>For capturing high-contrast&amp;nbsp; image data from an examined cell objects</td></tr><tr><td>Hydrophobic barrier pen (Elite PAP Pen)</td><td>Diagnostic Biosystems</td><td># K039</td><td>For defining cell perimeter for immunoflourescence staining</td></tr></tbody></table>

development and identification of a novel subpopulation of human neutrophil-derived giant phagocytes in vitro

中性粒细胞（PMN）是最适合他们的吞噬功能，对已知的入侵的病原体和微生物。它们具有最短半衰期之间的白细胞和中是组成性致力于凋亡它们的非激活状态。当招募到炎症部位来解决发炎，它们产生具有有效的抗微生物杀伤细胞毒性分子的阵列。然而，当这些强大的细胞毒性分子以不受控制的方式被释放它们可以破坏周围组织。然而，在最近几年，嗜中性粒细胞的多功能性日益证明，通过展示可塑性和免疫调节功能。我们最近发现了一种新的中性粒细胞衍生的亚群，其在标准培养条件下自发地发展不加入的细胞因子/生长因子如粒细胞集落刺激因子（GM-CSF）/白介素（IL）-4。他们残存的中性粒细胞吞噬能力，​​在很大程度上有助于增加他们的尺寸巨大;因此，他们分别命名巨噬细胞（Gφ）。与中性粒细胞，Gφ都长期生活在文化。他们表达的分化（CD），中性粒细胞标记CD66b / CD63 / CD15 / CD11b的/髓过氧化物酶（MPO）/中性粒细胞弹性蛋白酶（NE）集群，并且缺乏单核细胞谱系标志物:CD14 / CD16 / CD163和树突CD1C / CD141标记。他们还收乳胶和酵母多糖，并通过氧化爆裂与调理，酵母多糖和PMA回应刺激。 Gφ也表达了清道夫受体CD68 / CD36，不像嗜中性粒细胞，内在氧化低密度脂蛋白（脂蛋白）。此外，不同于新鲜嗜中性粒细胞，或培养的单核细胞，它们对oxLDL的摄取增加活性氧（ROS）的生产反应。此外，这些巨噬细胞含有微管相关蛋白-1轻链3B（LC3B）涂覆空泡，指示自噬的激活。使用特定的抑制剂显而易见的是，既吞噬作用和自噬是prerequisiTES为他们的发展和可能的NADPH氧化酶依赖的ROS。我们在这里描述为长寿命，中性粒细胞衍生的吞噬细胞在培养，其标识和其当前已知特征的这种新的亚群的制备方法。这个协议是用于获得并为了进一步调查它们的意义和功能表征Gφ必不可少的。

中性粒细胞自噬与发展文化嗜中性粒细胞自噬和它们在培养发展成Gφ7天内示于图3和4。由天4 - 7日，其规模是大大扩大，15和autophagosytosis是显而易见的，早在90分钟后共培养荧光染色膜（PKH-26，红色; PKH-67，绿色）的中性粒细胞。 14作为对照此嗜中性粒细胞亚群，一些嗜中性粒细胞的培养物也与GM-CSF / IL-4的处理。 14天的文化如前所述 - 大小于7细胞因子治疗的细胞增加。比Gφ18,19但是，体积较小并具有胞质突起类似的DC样细胞（ 图5），如先前b报道<html> Ÿ奥勒尔等。 19此外，GM-CSF / IL-4处理的细胞为阴性或具有低CD66b表达，15清楚地表明的形态和潜在的功能差异为好。 <img alt="图3" src="/files/ftp_upload/54826/54826fig3.jpg" /> 图3: 自噬在发展巨吞噬细胞（Gφ）的文化。新鲜分离的纯化的中性粒细胞标记PKH-67（绿色）或在零时间PKH-26（红色）膜荧光染料，然后共培养并随访至七天。细胞旋转到玻片上，细胞核用DAPI染色和样品通过共聚焦显微镜分析。自噬是已共培养90分钟后明显。红色和绿色到黄色和橙色合并在显影Gφ是显而易见的。文件/ ftp_upload / 54826 / 54826fig3large.jpg"目标="_空白"&gt; 点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54826/54826fig4.jpg" /> 图4: 巨型吞噬细胞（Gφ）在文化的发展。新鲜分离纯化嗜中性粒细胞随访至7天的文化。细胞在指定的时间间隔，与5月份的Grunewald，姬姆萨染色纺到玻片上，并用亮视野显微镜进行分析。少嗜酸性粒细胞的个体提出了比较。需要注意的是嗜酸性粒细胞的大小保持在培养不变。放大倍率100X油。 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54826/54826fig5.jpg" /> 图5: 巨吞噬细胞（Gφ） 和GM-CSF / IL 在培养处理的嗜中性粒细胞 发展的比较 。 （A）五月格伦沃尔德姬姆萨染色中性粒细胞无（Gφ）培养，并用GM-CSF / IL-4 7天。样品明亮的视野显微镜进行分析。放大，X40。在用补充有GM-CSF / IL-4的显示广泛胞质突起介质培养开发，但细胞比Gφ小。 （B）的新鲜分离的嗜中性粒细胞标记PKH-26（红色）染料和培养在无细胞因子的培养基中7天或用PKH-67（绿色）染料标记并培养在补充有GM-CSF / IL-4的用于中等7天。 1比率和共培养2小时:接着，开发了细胞在1混合。细胞固定并通过共聚焦MICR分析oscopy。这个数字已经从参考修改。 15 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了进一步研究Gφ发展的过程中，也再接时间推移显微镜的形态学变化。视频-1（第3天到第4天）和视频-2（第4天到第5天）表明在纯化的中性粒细胞培养物的发展。这些Gφ是非粘附或有限的运动能力轻易附着积极摄取中性粒细胞周围残余和碎片。在视频-3，单核细胞源性巨噬细胞和Gφ的运动在混合单核细胞/嗜中性粒细胞培养物相比较。该巨噬细胞活跃爬行（左，未标记的细胞）。该Gφ（右），是光明PKH-26标记的细胞。 视频1:演示巨噬细胞中纯化PMN文化上3天的发展- 4通过时间推移显微镜。中性粒细胞随访通过时间推移microscopy.The时间推移显微系统培养从第3天至4一天由电动倒置荧光显微镜，以及高分辨率/黑白CCD摄像头，与舞台上的孵化器。时间推移图像采集收购被带到每10分钟。最初发表于14引用<a href="https://www.jove.com/files/ftp_upload/54826/Video-1.mpg" target="_blank">请点击这里观看该视频。</a> <img alt="图1" src="/files/ftp_upload/54826/54826mov2.jpg" /> 视频2: -由钛5演示巨噬细胞中纯化PMN文化发展上4天我推移显微镜。 中性粒细胞随访通过时间推移显微镜文化从第4天至5一天。时间推移显微系统是由倒置电动荧光显微镜，和高分辨率/黑白CCD摄像机，与舞台上孵化器。时间推移图像采集收购被带到每10分钟。 <a href="https://www.jove.com/files/ftp_upload/54826/Video-2.mp4" target="_blank">请点击此处观看该视频。</a> <img alt="图1" src="/files/ftp_upload/54826/54826mov3.jpg" /> 视频3:巨噬细胞和共培养开发出了巨噬细胞。单核细胞/中性粒细胞共培养之后，上升时间推移显微镜从第4天至5一天。单核细胞来源的巨噬细胞（左）;亮（PKH-26染色细胞），中性粒细胞衍生的巨噬细胞（右）。用于视频的时间推移显微系统是由倒机动荧光MICR的的Oscope，和高分辨率/黑白CCD摄像机，与舞台上孵化器。时间推移图像采集收购被带到每10分钟。最初发表于14引用<a href="https://www.jove.com/files/ftp_upload/54826/Video-3.mp4" target="_blank">请点击这里观看该视频。</a> 标记在巨吞噬细胞表达 Gφ的嗜中性原点通过以下的中性粒细胞标记物CD66b / CD63 / MPO / NE / CD15（ 图6）的阳性表达验证。该Gφ还表示NADPH氧化酶，所述氧化低密度脂蛋白清道夫受体- CD68和CD36，并含有LC3B涂覆液泡和聚集物（通过Western印迹如LC3BII 15标识），这表明自噬标志物的存在。不过，他们均为阴性单核细胞谱系（CD14，CD16和CD163）和dendrit集成电路细胞（CD1C和CD141）标记物，这表明Gφ没有污染的单核细胞产生。 <img alt="图6" src="/files/ftp_upload/54826/54826fig6.jpg" /> 图6: 在培养7天为中性粒细胞，单核细胞和树突状细胞巨吞噬细胞（Gφ）各种标记的表达。中性粒细胞具体颗粒标记CD66b，嗜苯胺蓝粒标记CD63和MPO，弹性蛋白酶和CD15阳性表达。阴性表达对树突状CD1C和CD141标记和单核细胞系标记CD14，CD16和CD163。此外，Gφ表示，自噬标记LC3B的清道夫受体CD68和CD36和NADPH氧化酶亚基gp91-PHOX / P22-PHOX亚基。细胞核用DAPI染色，样品通过共聚焦显微镜分析。这个数字已经从引用修改。 14 </suP&gt;，15 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> Gφ的功能 - NADPH氧化酶激活，活性氧的产生和吞噬: 的乳胶珠吞噬和调理酵母在Gφ明显。 Gφ还产生固有ROS（ 图7A），以及由氧化爆裂（ 图7B-D）响应酵母多糖和PMA刺激。然而，与单核细胞或嗜中性粒细胞，Gφ产生的ROS还响应于氧化低密度脂蛋白的刺激和通过油红O（ 图7B中的F）进行染色。请注意，治疗与NADPH氧化酶抑制剂新鲜的中性粒细胞 - DPI，不仅抑制活性氧的产生，而且preventedGφ形成在培养物，这表明ROS信号传导对于Gφ形成至关重要。 14,15 <img alt="图7" src="/files/ftp_upload/54826/54826fig7.jpg" /> 图7: 氧化爆发，吞噬作用，并通过巨吞噬细胞吸收氧化低密度脂蛋白（Gφ）。 （ 一 ）基础ROS产生Gφ的溶酶体是显而易见的。 （ 二 ）ROS产生响应氧化LDL（氧化低密度脂蛋白），PMA和酵母多糖（酵母多糖颗粒明确指出）。在Gφ（C）硝基四氮唑（NBT）测试显示了不和PMA（幻灯片是未染色的，但刀片与五月格伦沃尔德，姬姆萨染色）呼吸爆发活动。 （D）NBT测试，并且可格鲁内瓦尔德，姬姆萨染色Gφ与PMA和PMA / DPI其抑制NADPH氧化酶和活性氧。升（E）吞噬ATEX和IgG调理酵母在PKH-26（红色）染色的细胞。在未处理和氧化低密度脂蛋白（F）油红O染色处理Gφ。这个数字已经从引用修改。 14，15 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 中性粒细胞轮回跨内皮细胞为了确定潜在的嗜中性粒细胞亚群可能发展成Gφ，嗜中性粒细胞，通过血管内皮细胞单层迁移测定（ 图8A）。 90分钟后，将嗜中性粒细胞的62.3±12.2％，在下部隔室通过内皮细胞transmigrated向IL-8的。值得注意的是，Gφ正面为只从嗜中性粒细胞，而从该非迁移的中性粒细胞级分开发的细胞的transmigrated人口开发CD66b / CD15 / LC3B均大小为嗜中性的标记更小和负CD66b / CD15（ 图8B，8C） 。 <img alt="图8" src="/files/ftp_upload/54826/54826fig8.jpg" /> 图8: 通过对巨噬细胞（Gφ）形成的内皮细胞IL-8依赖的中性粒细胞轮回的影响。 （ 一 ）对说明IL-8跨内皮细胞单层（ECS）的中性粒细胞轮回试验方案。该测定可以被视为对嗜中性粒细胞募集到急性炎症部位的模型。 （B - C）在细胞迁移测定（在协议3规定），transmigrating（B）和非迁移（C）的嗜中性粒细胞压裂系统蒸发散七天无生长因子中培养（如协议1）。然后，将细胞旋涂到载玻片并通过共聚焦显微镜分析。固定的细胞进行染色CD66b（红），LC3B（绿）和CD15（红）。细胞核用DAPI（蓝色）染色。 <a href="http://ecsource.jove.com/files/ftp_upload/54826/54826fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro at JoVE.com

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

我们在这里描述的获取和识别中性粒细胞衍生的巨噬细胞的新特点亚群的方法。这些细胞发展从新鲜人血中性粒细胞培养，并通过吞噬，吞噬，非常大的规模，并延长寿命的特点。这种方法是必不可少的进一步调查这一独特的中性粒细胞衍生的细胞亚群。

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 ...

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Immunology and Infection

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

15.1K 次观看.  Technion-Israel Insitute of Technology. 我们在这里描述的获取和识别中性粒细胞衍生的巨噬细胞的新特点亚群的方法。这些细胞发展从新鲜人血中性粒细胞培养，并通过吞噬，吞噬，非常大的规模，并延长寿命的特点。这种方法是必不可少的进一步调查这一独特的中性粒细胞衍生的细胞亚群。 

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

科研

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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

生物工程

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

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

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

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

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

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

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.

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

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

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 ...

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群

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此视频中的章节

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

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

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

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

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

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

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

一套快速了解中性粒细胞功能的筛查技术