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>

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			<td height="20" style="height:20px;">Giemsa stain Kit</td>
			<td>Sigma</td>
			<td># 48900</td>
			<td>Cell biololgy grade-(procedure No GS-10)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BioIndustries</td>
			<td># 03090105</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Human Interleukin-8 (CXCL8)</td>
			<td style="width:231px;">PeproTech</td>
			<td># 200-08-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD14 (clone 5A3B11B5)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td># sc-58951</td>
			<td>Mouse IgG2b; expressed by&#160; monocytes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Anti-CD66b (clone 80H3)</td>
			<td>AbD Serotec</td>
			<td># MCA216</td>
			<td>Mouse IgG1; expressed by neutrophils</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD1c (BDCA-1) (clone AD5-8E7)</td>
			<td>MACS Miltenyi Biotec</td>
			<td># 130-090-695</td>
			<td>Mouse IgG2a; expressed by&#160; dendritic cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD15 (clone MY-1)</td>
			<td>Abcam</td>
			<td># ab754</td>
			<td>Mouse IgM; expressed by neutrophils</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Anti-CD68</td>
			<td>Protein Tech</td>
			<td># 16192-1-AP</td>
			<td>Rabbit IgG; to recognize oxLDL scavenger receptor&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-LC3B</td>
			<td>Sigma</td>
			<td># L7543</td>
			<td>Rabbit IgG&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myeloperoxidase</td>
			<td>Abcam</td>
			<td># ab45977</td>
			<td>Rabbit IgG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Neutrophil elastase</td>
			<td>Calbiochem</td>
			<td># 481001</td>
			<td>Rabbit IgG&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-NOX2 (gp91-phox)</td>
			<td>Abcam</td>
			<td># ab131083</td>
			<td>Rabbit IgG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD36 (SR-B3)</td>
			<td>Novus Biologicals</td>
			<td># NB400-144</td>
			<td>Rabbit IgG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Purified Mouse IgG1, &#954; Isotype Control (clone MG1-45)</td>
			<td>BioLegend</td>
			<td># 401401</td>
			<td>Antibody used as isotype control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Purified Mouse IgG2a, &#954; Isotype Control (clone MOPC-173)</td>
			<td>BioLegend</td>
			<td># 400263</td>
			<td>Antibody used as isotype control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal rabbit IgG</td>
			<td>Santa Cruz Biotechnologies</td>
			<td># sc-2027</td>
			<td>Antibody used as isotype control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CF488A Goat Anti-Rabbit IgG (H+L)</td>
			<td>Biotium&#160;</td>
			<td># 20012</td>
			<td>Anti-Rabbit IgG with the green fluorescent dye CF488A</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CF647 Goat Anti-Rabbit IgG (H+L)</td>
			<td>Biotium&#160;</td>
			<td># 20043</td>
			<td>Anti-Rabbit IgG with the red fluorescent dye CF647</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CF488A Goat Anti-Mouse IgG (H+L)</td>
			<td>Biotium&#160;</td>
			<td># 20010</td>
			<td>Anti-Mouse IgG with the green fluorescent dye CF488A</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CF647 Goat Anti-Mouse IgG (H+L)</td>
			<td>Biotium&#160;</td>
			<td># 20040</td>
			<td>Anti-Mouse IgG with the red fluorescent dye CF647</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent Mounting Medium with DAPI</td>
			<td>Vectashield H-1000; Vector Lab Inc.</td>
			<td># E19-18</td>
			<td>Nuclear staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal laser scanning microscope (LSM 700)&#160;&#160;&#160;</td>
			<td style="width:231px;">Carl Zeiss</td>
			<td>Ser.# 3523000380</td>
			<td>Plan Apo x40 immersion oil objective&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss CLSM software (ZEN 2010)</td>
			<td style="width:231px;">Carl Zeiss MicroImaging GmbH</td>
			<td>version 6.0</td>
			<td>For colocalization analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">ImageJ software</td>
			<td style="width:231px;">Wayne Rasband, NIH, USA</td>
			<td style="width:131px;">version 1.49k</td>
			<td>For determination of cell&#160; areas and fluorescence intensity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Light microscope (Axiovert 25)</td>
			<td style="width:231px;">Carl Zeiss</td>
			<td>Ser.# 201060153</td>
			<td>Examination of cells in culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Centrifuge (Megafuge 1.0 R)</td>
			<td style="width:231px;">Heraeus Instruments</td>
			<td># D-37520</td>
			<td>Cells separation from blood; cytospins preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted fluorescent microscope (Zeiss Axio Observer Z.1)</td>
			<td style="width:231px;">Carl Zeiss</td>
			<td>Ser.# 3834001470</td>
			<td>Demonstration of giant phagocytes development</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Temperature-controlled incubation system (Cube&#38;Box)</td>
			<td>Life Imaging Services</td>
			<td style="width:131px;"></td>
			<td>Temperature control system for microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">High&#160;resolution digital CCD camera (AxioCam HRm)</td>
			<td style="width:231px;">Carl Zeiss</td>
			<td>Ser.# 117090279</td>
			<td>For capturing high-contrast&#160; image data from an examined cell objects</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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Research

JoVE Journal

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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免疫与感染

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间:应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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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