This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

1.中性粒细胞分离共培养原发性白血病细胞注:过程是里昂医院伦理委员会与签署知情同意所有患者同意下进行的。 <ol><li> 原代白血病细胞和中性粒细胞的分离 <ol><li>从确诊为慢性淋巴细胞白血病（CLL）的患者收集EDTA（每毫升血液1.8毫克EDTA）外周血管。 </li><li>各15毫升的血液添加到无菌的50ml管中，并用15毫升的RPMI稀释（稀释1:1），然后小心地慢慢加入溶于15毫升的密度梯度溶液到管的底部不混合各相。确保该密度梯度的溶液是在室温下的制备。 </li><li>离心机300 XG 35分钟，在室温和不带刹车。血液应该分成四个不同的阶段， 如图1A所示 ，从上到下依次为:血小板和血浆，单核细胞（重量海特环），密度梯度液，粒细胞和红细胞。 </li><li>收集其表示原代白血病细胞中，用塑料巴斯德吸管，并转移到一个新的50ml管中的白圈。 <ol><li>填用PBS管（包含钙和镁）至总体积为50ml，并离心以300g在室温10分钟。 </li><li>重悬沉淀用5毫升的PBS再向上总和离心机以300g添加的PBS至50ml在室温10分钟。 </li><li>用完全RPMI培养基重悬沉淀（RPMI 1640补充有10％胎牛血清（FBS），2mM谷氨酰胺，100U / ml青霉素和100mg / ml链霉素），用于使用细胞生存力计数器计数。 </li></ol></li><li>抽吸上部相离开粒细胞和红细胞相和加起来的PBS至25ml，然后在0.9％NaClup添加3％葡聚糖至总体积为50ml。混合管10次，并在室温保持他们30分钟没有混合。 </li><li>收集上RBC贫中性粒细胞层的d将它们放入一个干净的无菌50ml管中，并以300g离心管在室温下10分钟。重悬每片用5毫升的PBS然后总加起来红细胞裂解液至50ml。 </li><li>保持管在黑暗中进行15分钟，在室温，然后在500g下离心在RT 10分钟。用PBS洗粒料（加起来PBS中至总体积为50ml），然后在500g下离心在RT 10分钟。 </li><li>悬浮用完全RPMI培养基中的小球用于使用细胞生存力计数器计数。保持每个细胞类型的3×10 5 5毫升塑料管50微升的PBS-FBS（4％），以确定使用流式细胞术其隔离的纯度。 </li></ol></li><li> 分析分离细胞种群形态外观 <ol><li>这样做，重新悬浮每3×10 4个嗜中性粒细胞，并在150微升完全RPMI培养基3×10 4个单核细胞。组装载玻片，滤波器卡，并在细胞离心涂片离心机样品室，确保吨他离心机是很好的平衡。 </li><li>将不同的细胞类型到样品室和CYTO-离心在室温750 XG 10分钟从离心机中取出的幻灯片，过滤器卡和样品室。仔细拆解，以免损坏幻灯片上的细胞。丢弃过滤器卡和样品室。 </li><li>染色使用吉姆萨染色试剂盒的细胞和保持载玻片1小时干燥。通过用放大100倍的显微镜检查细胞。 </li></ol></li><li> 测试用流式细胞仪分离的细胞纯度 <ol><li>标记的分离的原代白血病细胞用缀合APC的抗人CD19抗体（5微升/ 10 6个细胞）。与表1（5微升/ 10 6个细胞）中列出的抗人抗体的混合物标记纯化的中性粒细胞。孵育在黑暗细胞30分钟，在4℃ </li><li>清洗细胞用500μl的PBS-FBS（4％）离心机以300g在室温5分钟的管中。 </li><li>重悬在200μl的PBS-FBS（4％）每个粒料。 </li><li> 488纳米激光:FSC-A（488纳米），SSC-A（488 / 10BP），FITC（530/30 BP），PerCP-经Cy5.5（40分之695BP流式细胞仪上使用下面的光学结构流分析），PE（575/26 BP）; 633纳米激光:APC（660/20 BP），APC-Cy7的（780/60 BP）。确保色彩补偿。 </li></ol></li><li> 自体原发性中性粒细胞白血病细胞共培养 <ol><li>种子2×10 5个细胞/ ml单独使用或与在完全RPMI培养基中1:10自体的中性粒细胞初级白血病细胞。这样做，通过稀释细胞悬浮液调整细胞数和2×10 5个细胞/ ml初级白血病细胞添加到2×10 6个细胞/ ml的中性粒细胞。仔细混合。 </li><li>添加布鲁顿的酪氨酸激酶（BTK）抑制剂（ibrutinib）为25μm到细胞中。用5％的CO 2下孵育24小时，在37℃。 </li></ol></li><li> 细胞收集和分析FACS <ol><li>科尔等将细胞在5ml塑料管用于FACS分析和离心机以300g在室温5分钟的管中。用1ml的PBS-FBS（4％）洗涤粒料然后以300g离心在RT 5分钟。 </li><li>在膜联蛋白V重悬粒料和PI使用商业试剂盒和孵化在黑暗中的细胞在室温10分钟。 488纳米激光:上仪使用图2A的门控策略的流程和以下光学结构分析的FSC-A（488纳米），SSC-A（488 / 10BP），FITC（530/30 BP）的PI（610 / 20 BP）。确保色彩补偿。 </li></ol></li></ol> 2.沿粒途径及其与三维模型RL淋巴瘤B细胞共培养HL60细胞分化<ol><li> 人早幼粒（HL60）细胞分化为中性粒细胞样细胞 <ol><li>调整HL60细胞数至3×10 5个细胞/ ml，然后添加1μM的视黄酸（RA）和1.25％DMSO中，诱导其分化。种子每3×10 5个 </s向上&gt;每孔在48孔板HL60细胞，然后在37℃用5％CO 2孵育48小时。 </li><li>后，收集细胞并离心以300g在室温5分钟。悬浮用完全RPMI培养基中的细胞，使用细胞生存力计数器计数。 </li><li>然后稀释细胞悬浮在完全RPMI培养基中以调节HL60细胞数至3×10 5个细胞/ ml，并通过加入1μM的视黄酸（RA）和1.25％DMSO的再次诱导其分化。种子每3×10 5 HL60细胞每孔在48孔板中并在37℃用5％CO 2孵育另外48小时。 </li></ol></li><li> HL60分化分析（HL60 差异 ） <ol><li>收集细胞，并以300g离心管在RT 5分钟。重悬完全RPMI培养基颗粒使用细胞活力计数器计数。 </li><li>分析通过流式细胞术在细胞表面标志物的表达的变化。将每个3×10 5个 </suP&gt;未分化的HL60细胞（来自细胞培养物）并在5ml塑料管用于FACS分析和离心机以300g在室温5分钟管3×10 5 HL60 差异细胞。 </li><li>重悬在50μl的PBS-FBS（4％）的粒料。标签用抗人CD11b的缀合AF700或缀合APC的抗人CD38（5微升/ 10 6个细胞）的细胞。孵育在黑暗细胞30分钟，在4℃ </li><li>用500μl的PBS-FBS（4％）洗涤，然后以300g离心在RT 5分钟。重悬在200μl的PBS-FBS（4％）的粒料。 </li><li>对流动分析仪使用图3A的门控策略及以下的光学配置:488 nm激光:FSC-A（488纳米），SSC-A（488 / 10BP）; 633纳米激光:APC（660/20 BP）的Alexa Fluor 700（730/45 BP）。 </li><li>为了测试在HL60 差异的形态变化，细胞固相化到玻片上进行显微镜检查。 <ol><li>这样做，重新悬浮每3×10 <suP&gt; 4未分化的HL60细胞（来自细胞培养物）和3×10 4的HL60 差异在150微升完全RPMI培养基。组装载玻片，滤波器卡，以及样品室中的细胞离心涂片离心机，确保离心机是平衡的。 </li><li>将每个细胞类型到样品室和CYTO-离心机以1,500rpm在室温10分钟。取出幻灯片，滤波器卡，并从离心机样品室。仔细拆解，以免损坏幻灯片上的细胞。丢弃过滤器卡和样品室。 </li><li>染色使用吉姆萨染色试剂盒的细胞和保持载玻片1小时干燥。通过用放大100倍的显微镜检查细胞。 </li></ol></li></ol></li><li> 三维（3D）培养 注:确保在本实验中所用的材料是冷和实验正在发生在冰上。 <ol><li>悬浮每5×10 4的RL细胞，单独或与HL60 差异混合</em&gt;在比RL:HL60 差异 1:10使用，以扩大所述开口至约2至3毫米的无菌剪刀切1毫升枪头300μl的基底膜基质。在此步骤期间避免气泡。 </li><li>种子各300微升细胞悬浮液/孔在24孔板中。在此步骤期间避免气泡。温育30分钟的板在37℃，5％的CO 2然后加入1ml完全RPMI培养基每个孔，并在37℃用5％CO 2孵育7天。改变介质每两天。在第5天加10纳米长春新碱。 </li></ol></li><li> FACS分析 <ol><li>经过7天培养的，吸出培养基并用1ml冰冷的PBS洗每个孔两次。加3毫升/冰冷的PBS-EDTA（5毫米）的井。通过使用200微升枪头的底部刮分离从井的底部的凝胶。轻轻摇动板在冰上30分钟。 </li><li>转移细胞悬液到无菌15ml试管并轻轻摇晃在IC管E对于30分钟。检查同质细胞悬浮液的外观。 （如果不是的话，那么摇晃细胞较长时间或添加更多的PBS-EDTA）。 </li><li>离心管，在300×g离心在RT 10分钟。用PBS洗粒料然后在300 xg离心离心在RT 10分钟。 </li><li>用PBS-FBS（4％）重悬，并与缀合PE-Cy7的和缀合APC的抗人CD38抗体（5微升/ 10 6个细胞）的抗人CD19抗体标记。孵育在黑暗的30分钟，在4℃ </li><li>用500μl的PBS-FBS（4％），然后离心管洗以300g在室温5分钟。重悬以膜联蛋白V和PI使用商业试剂盒的细胞。 </li><li>对流动分析仪使用图4A的门控策略和下述光学配置:488纳米激光:FSC-A（488纳米），SSC-A（488 / 10BP），FITC（530/30 BP）的PI（610 / 20 BP），PE-Cy7的（780/60 BP）; 633纳米激光:APC（660/20 BP）。确保色彩补偿。 </li></ol></li&gt; </ol>

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J. Cancer Oxf.Engl 1990. 45, 1950-1958 (2009).</li><li>Zhu, Q., 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=The+IL-6-STAT3+axis+mediates+a+reciprocal+crosstalk+between+cancer-derived+mesenchymal+stem+cells+and+neutrophils+to+synergistically+prompt+gastric+cancer+progression.">The IL-6-STAT3 axis mediates a reciprocal crosstalk between cancer-derived mesenchymal stem cells and neutrophils to synergistically prompt gastric cancer progression.</a> Cell Death Dis. 5, 1295 (2014).</li></ol>

The authors have nothing to disclose.

这里所描述的，有效的，简单，快速和廉价的协议为嗜中性粒细胞的人血液中分离，使用密度梯度离心的方法的高纯度和相同的步骤的单核细胞内也分离和回收。所述分离的细胞群是≥90％的纯度。 有几种方法可用于从人血液嗜中性粒细胞的隔离。这些包括类似的方法使用连续梯度11,12，或使用嗜中性粒细胞分离试剂盒由阳性免疫磁性选择期间的中性粒细胞是免疫磁性与特异性抗体标记的诸如抗人CD16然后嗜中性粒细胞通过CD16-结合富集的在磁性柱13阳性的细胞。负免疫磁性选择商业套件也可在此期间，全血或粒细胞悬液被标记与细胞上的其他塔结合的抗体的鸡尾酒Ñ ​​中性粒9（ 即，标记红细胞，血小板和不想要的细胞，如CD2，CD3，CD9，CD19，CD36，CD56，血型糖蛋白A和葡聚糖包被的磁性颗粒的抗体复合物），然后嗜中性粒细胞被洗脱富集的抗体不在磁性柱结合的细胞的阴性部分。此外，嗜中性粒细胞可以通过荧光激活细胞分选14分离。 一些技术已经显示出提供纯嗜中性粒细胞，如阳性免疫磁性选择的高产量。但是，这种方法有缺点相比，由于结合嗜中性粒细胞的表面上，这可能通过诱导其激活或分化改变它们的功能的标记试剂密度梯度离心的方法。还使用正免疫磁性选择方法，在用于分离的磁列的抗体标记的嗜中性粒细胞结合，这也可能会影响其功能。荧光激活细胞分选具有另一限制，只要该方法需要较长的时间来收集可能嗜中性粒细胞存活产生不利影响的细胞由于它们的短半衰期。 密度梯度离心法是非常相当和嗜中性粒细胞的纯度可以使用这两个过程超过90％，但前者是一个两层梯度相比其中包括40％，60％和80％（体积）成层梯度溶液这在技术上是较少有挑战性/体积的PBS。有人提到由Swamydas等人15的梯度溶液界面的相互混合经常发生由于三层之间的小的密度差异。相对于该负免疫磁性选择方法，它们也有报道是有效的高纯度和生存能力9嗜中性粒细胞的隔离。他们在积极的免疫磁性选择和荧光激活细胞分选的优点是中性粒细胞未标记的任何标记试剂和不结合磁性柱，从而避免了细胞的活化，但这种方法是昂贵得多多的时间相比，密度梯度法耗时。 嗜中性粒细胞通常经历快速自发凋亡在体外和体内 16,17。 HL60promyelocyticcells保留人类白细胞祖细胞的许多特性，如经过分化成嗜中性粒细胞18的潜力。与中性粒细胞，HL60 差异细胞不迅速凋亡，并使用这些细胞有解决的嗜中性粒细胞的反应不同的麻烦，由于来自不同供体的隔离。 最近，3D培养变得更加成熟和相关的人类和动物生理学，特别是在癌症领域对于不同的生物研究一个有吸引力的模型。从2D模式转变到3D的文化进展迅速州市NCE后者是学习的各种病理疾病如癌症19中更加显着。人们越来越2D文化弊端的认识，其中增加了第三维细胞的环境，以便采取在细胞相互作用20的重要性，定睛一看，来研究细胞行为和特点的差异非常重要。 3D培养产生导致更多的相关研究类似于在活生物体的环境条件（ 例如 ，人类）。然而，3D文化涉及不同的合作伙伴，如细胞，细胞外基质和间质液哪些不存在于2D文化之间复杂的相互作用。因此，将努力以建立方法校准和对优良实验室规范以及有效供给尤其是商业品牌的广泛测试和监控计数需要。 肿瘤微环境由多种细胞类型，包括参与和调节肿瘤发生，转移和应对抗癌药物21,22多种免疫细胞群。几项研究已经表明，在肿瘤中的增加中性粒细胞浸润的是显著与获得性抗性相关的几个抗癌剂诸如抗VEGF治疗23,24。另外，仰角在预处理嗜中性粒细胞在治疗转移性肾细胞癌25，以及高水平的患者不同实体瘤肿瘤内嗜中性粒细胞的存在数，26已被提议作为生存预后不良因素。我们的研究表明，嗜中性粒细胞可以在保护淋巴瘤B细胞从抗癌剂诱导的细胞凋亡中发挥作用。 总之，这里所描述的可靠和可再现的方法可通过任何实验室被用来收集来自嗡嗡声中性粒细胞和单核细胞的血。此外，沿粒通路HL60细胞的分化，以避免一些neurophil问题，提出诸如迅猛自发凋亡。这些方法被用来研究对淋巴瘤细胞的抗淋巴瘤剂的灵敏度的中性粒细胞的作用，其中的中性粒细胞上展示针对利用二维和三维培养系统这些试剂淋巴瘤细胞有保护作用。这些方法可用于多种嗜中性粒细胞的功能性研究应加深我们在肿瘤生物学中性粒细胞的作用的理解。特别是该方法可用于更好地理解中性粒细胞和肿瘤细胞之间或中性粒细胞和微环境的其他组件之间的串扰。

先天免疫细胞构成的细胞的肿瘤微环境内的重要比例，并已与患者和癌症1动物模型中的肿瘤恶性相关。最近，已变得更加广泛地理解，慢性免疫应答中促进肿瘤进展，转移和耐化学疗法2发挥关键作用。巨噬细胞是已被证明直接调节对化疗3,4-肿瘤细胞应答重要的先天免疫细胞。然而，中性粒细胞，在先天免疫系统的主要参与者的作用，在调节肿瘤响应于抗癌治疗是不知道。这些协议的目的是使用快速和可靠的方法来分离来自CLL患者的血液样本的中性粒细胞和分化的HL60细胞沿粒细胞途径，以研究在调节淋巴瘤细胞抗淋巴瘤剂的敏感性的作用。 6在血液5，并作为防御的第一道防线是先天免疫系统的最丰富的细胞成分。嗜中性粒细胞有几种病理条件7上升除了可变效应子功能有效先天免疫反应中起重要作用。因此，以分离从其它血细胞，如密度梯度分离法中性粒细胞的快速和可靠的方法，需要在体外研究。使用这种方法的中性粒细胞隔离将有利于在体内和体外中性粒细胞介导的免疫功能进一步研究。 以获得嗜中性粒细胞的纯群体的能力对于患者的调查与免疫疾病8的重要的第一步。密度梯度分离方法是在其中获得的细胞高产率的理想的技术。该方法具ð涉及在含有稀释的人血，然后离心以300g为无休息35分钟试管底部加入密度梯度溶液中。的单核细胞的环出现在界面和嗜中性粒细胞驻留低于前者。这种方法具有相对于其他可用的方法显著优点，如嗜中性粒细胞分离试剂盒这是更昂贵的9。此外，通过使用针对特异于人嗜中性粒细胞的表面标志物的抗体的商品试剂盒中分离人血液嗜中性粒细胞，增强细胞活化或分化的风险。密度梯度分离方法允许嗜中性粒细胞的一个短的时间周期内的隔离。在同一步骤，单核细胞也分离和回收。它是在其中，以实现功能完整性得到纯的细胞的高收率的基本技术。 为了模仿肿瘤microenvironment，进行3D实验。定体外中性粒细胞的半衰期很短，用新鲜的人中性粒细胞的3D实验不是决定性的。出于这个原因，早幼粒细胞（HL60）细胞被诱导分化成使用分化诱导剂二甲基亚砜（DMSO）和视黄酸（RA）嗜中性粒细胞样细胞。用分化的HL60细胞（HL60 差异 ）将防止有中性粒细胞，由于来自不同供体隔离不同的反应。 体外三维培养模型代表在体外 2D模型之间和体内模型中的中间阶段。在二维培养，细胞扩散塑料表面上形成不自然的细胞附件被这个合成表面沉积变性的蛋白质。相反，在由于细胞和它们合成的细胞外基质的3D培养形式自然细胞 - 细胞附件细胞对它们所连接的天然材料。出于这个原因，3D共培养模型，尤其是癌症细胞和其他细胞类型之间，已经指示他们的肿瘤的生长，血管生成和转移的贡献非常有用的。其结果是，3D培养使细胞培养模仿存在于体内 10的生理条件。

<table border="0" cellpadding="0" cellspacing="0" width="1161">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">RPMI 1640&#160;</td>
			<td style="width:157px;">Gibco Invitrogen</td>
			<td style="width:156px;">21875-034</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td>
			<td>Life technologies</td>
			<td style="width:156px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin (Pen Strep)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruton&#39;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&#160;</td>
			<td style="width:157px;">CliniSciences</td>
			<td style="width:156px;">A3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine&#160;</td>
			<td style="width:157px;">EG labo</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Red cell lysis buffer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:156px;">555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll (Pancoll)</td>
			<td>PAN Biotech</td>
			<td style="width:156px;">P04-60500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Retinoic acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine serum albumin (BSA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>A7906</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">E5134</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium chloride (NaCl)</td>
			<td>Euromedex</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexing V-FLOUS staining kit&#160;</td>
			<td style="width:157px;">Roche</td>
			<td>11&#160;988&#160;549&#160;001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kit RAL 555 Modified Giemsa staining kit</td>
			<td style="width:157px;">Cosmos Biomedical</td>
			<td style="width:156px;">CB361550-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cytocentrifuge</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMR-XA microscope</td>
			<td style="width:157px;">Leica Microsystems</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cellometer Auto T4 Cell Viability Counter</td>
			<td style="width:157px;">Nexcelom&#160; Bioscience</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

这里所描述的密度梯度分离方法提供了从CLL患者的血液中分离的原代白血病细胞和未刺激的嗜中性粒细胞图1A表示密度梯度离心后获得的不同血液层（从上到下:血小板和血浆，白圈表示的单核细胞，密度梯度溶液，粒细胞和红细胞）。 图1B和1C分别示出嗜中性粒细胞（多叶形核细胞）和单核细胞之间的形态出现的差异。 结果如图1D表示前向散射（FSC）与原白血病细胞（单核细胞）的侧散射（SSC）的情节。该标签与人口共轭APC抗人CD19显示直方图（ 图1E）的只有一个完整的右移高峰这表明这类人群积极为CD19和纯净。嗜中性粒细胞也≥90％纯度，与表1中所列的荧光染料缀合的单克隆抗体的混合物标记的分离的嗜中性粒细胞后流式细胞流动验证。 图1F表示FSC VS分离的嗜中性粒细胞这是阳性CD45的SSC散点图（ 图1G），积极为CD15和阴性CD14（ 图1H），积极为CD15和CD16（ 图1I），积极为CD16和阴性CD56（ 图1J）。 <img alt="图1" src="/files/ftp_upload/53846/53846fig1.jpg" /> 图1.分析形态表现及初级白血病细胞纯度和中性粒细胞中分离出患者的血液中。 （A）的示意图代表密度gradi后不同血型层耳鼻喉科离心。（BC）吉姆萨染色代表（B）的嗜中性粒细胞和（C）原代白血病细胞（单核细胞）之间的形态差异。照片是通过用放大100倍的显微镜（D）前向散射（FSC）和侧散射（SSC）曲线表示的分离的原代白血病细胞群（E）分离的原代白血病细胞用抗CD19-APC和分析CD19的表达。红色线表示未标记的细胞和蓝色线表示标记的抗CD19-APC的细胞。（F）的 FSC VS SSC散点图表示分离的嗜中性粒细胞群。（GJ）隔离嗜中性粒细胞标记与表中列出的抗体的混合物1和（G）的 CD45，（H）的 CD14和CD15，（Ⅰ）CD15和CD16，（J），CD16的表达进行分析ðCD56。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53846/53846fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 抗原 </td><td> 荧光 </td><td> 克隆 </td></tr><tr><td> CD14 </td><td> APC-Vio770（CY-7） </td><td> TÜK4 </td></tr><tr><td> CD15 </td><td> APC </td><td> VIMC6 </td></tr><tr><td> CD16 </td><td> FITC </td><td> VEP13 </td></tr><tr><td> CD45 </td><td> PerCP-Vio700（经Cy5.5） </td><td> 5B1 </td></tr><tr><td> CD56 </td><td> PE </td><td> AF12-7H3 </td></tr></tbody></table> 表1.荧光标记的纯化的单克隆抗体用于测试隔离中性粒细胞的纯度。从美天旎生物技术获得所有抗体。 <p class="jove_content"FO:保持-together.within页="1"&gt;为了研究嗜中性粒细胞的初级白血病细胞（CLL）到ibrutinib的灵敏度的效果，后者被单独使用或与自体的中性粒细胞（N）的24小时在比N培养:CLL 10:1，在存在或不存在ibrutinib在25微米。活原代白血病细胞（膜联蛋白V阴性/ PI阴性）的比例通过用膜联蛋白V-FITC和PI双染色，随后通过流式细胞术分析测定。 FSC VS在图2A的SSC散点图表示嗜中性粒细胞和原代白血病细胞的栅极。中性粒细胞是更细化比主白血病细胞，使他们出现高SSC。对原代白血病细胞中ibrutinib存在嗜中性粒细胞共培养选通，结果表明活细胞的比例更高（ 图2C，84.8％），相比于单独培养的细胞（ 图2B，53.1％）。在图2D的方块图显示了增减通过ibrutinib诱导细胞活力电子中性粒细胞的存在（40.1±3.1 VS 60.1±3.5，P &lt;0.001，19例）显著抑制。 <img alt="图2" src="/files/ftp_upload/53846/53846fig2.jpg" /> 图2.自体中性粒细胞防范Ibrutinib。血主白血病细胞是从诊断为慢性淋巴细胞白血病（CLL）的患者收集。初级白血病细胞中分离，并与在原代白血病细胞自体的中性粒细胞（N），单独或一起培养:Nratio 1:10 24小时，在存在或不存在25μM的ibrutinib的。活原代白血病细胞的百分比（Annexiv V负/ PI阴性）通过用膜联蛋白V-FITC和PI双染色，随后通过流式细胞术分析测定（A）中前向散射（FSC）和侧散射（SSC）曲线表示嗜中性粒细胞和原代白血病细胞中的栅极（B）的双维斑点印迹显示了单独培养，并用25微米ibrutinib治疗活和凋亡初级白血病细胞的百分数（C）双维斑点印迹显示了活和凋亡初级白血病细胞的百分比CO-与嗜中性粒细胞培养，并用25微米ibrutinib处理。（D）的方框的曲线表示的19名患者存活原代白血病细胞的百分比。数据表示为平均值±标准差。 *** P &lt;0.001 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53846/53846fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 嗜中性粒细胞在体外的半衰期很短，使它们在三维培养使用无效。沿粒途径HL60细胞分化诱导分化诱导剂。两个不同的参数（细胞表面标记表达和形态学昌ES）被用来测​​量HL60分化。 FSC VS在图3A中的SSC散点图表明，HL60细胞是相比HL60 差异细胞具有较高的FSC的尺寸较大。标记与缀合至AF700和缀合APC的抗人CD38antibody抗人CD11b抗体的细胞（HL60或HL60 差异 ）显示在分化的增加CD11b和CD38的表达（ 图3B），这是一个指示性粒细胞的分化。 HL60 差异细胞还显示了由多叶形核（ 图3C）的外观检测形态的变化。 <img alt="图3" src="/files/ftp_upload/53846/53846fig3.jpg" /> 图3.分化的HL60细胞的结构参数。 （A）前向散射（FSC）和侧散射（SSC）图表示HL60和差异化的HL60细胞（HL60 差异 ）。（B）HL60和HL60 差异细胞用抗CD11b-AF700或抗CD38-APC，随后通过流式细胞术分析。门控在FSC-A与SSC-A散点图（A）每个细胞群后，分析细胞的细胞CD11b或CD38的表达。（C）HL60细胞表现出朝向粒细胞分化一致的形态变化。照片是采取显微镜放大100倍。大胆的黑色箭头指向多裂核。图代表了五个独立的实验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53846/53846fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 以测试调节到在三维模型长春新碱RL细胞应答HL60 差异的细胞的作用，RL细胞单独或与在10中的n在长春新碱的存在或不存在基底膜基质的HL60 差异培养M.使用图4A所示的门控策略，既种群都基于CD19和CD38的表达鉴别; RL阳性细胞CD19和HL60 差异阳性细胞CD38。关于RL细胞与长春新碱的存在下HL60 差异细胞共培养的选通，结果表明活细胞的比例更高（图4C中，35.9％），相比于单独培养RL细胞（ 图4B中 ，19.7％）。在图4D的条形图显示了在活RL细胞的百分比在HL60 差异细胞（19.5±0.2 VS 33.1±1.0，P &lt;0.001）的存在下，显著增加（CD19阳性膜联蛋白V阴性/ PI阴性）。 <img alt="图4" src="/files/ftp_upload/53846/53846fig4.jpg" /> 图4:中性粒细胞样HL60 差异 细胞防范长春新碱RL淋巴瘤细胞三维培养。RL细胞<html>用HL60 差异细胞以RL单独或一起培养:HL60 差异比1:10 7天在基底膜基质。在第5天，长春新碱（VCR）在10nM的浓度加入。球状体解离第7天与细胞用抗CD19-PECy7和抗CD38-APC，然后用膜联蛋白V-FITC和PI随后通过流式细胞术分析再悬浮（A）正向散射（FSC）和侧散射（SSC）曲线表示RL细胞和HL60 的diff单元的栅极。（B）的双维斑点印迹显示了单独培养，并用10nM长春新碱治疗活和凋亡RL细胞百分比。（C）的双维点状印迹显示了活和凋亡RL细胞HL60 差异共培养并用10nM长春新碱治疗的百分比（D）的条形图表示活RL细胞的百分比。数据表示为平均值±标准差。 *** P≤0.001。jove.com/files/ftp_upload/53846/53846fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

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

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 ...

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.2K 次观看.  Cancer Research Center in Lyon. 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. 

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

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

科研

免疫与感染

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

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

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

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

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

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

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

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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 ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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 ...