Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00.000+00:00
Duration: 14 min 4 s
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

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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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 3, 309-322 (2012).</li><li>Shojaei, F., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+to+antivascular+endothelial+growth+factor+treatment:+role+of+myeloid+cells.">Refractoriness to antivascular endothelial growth factor treatment: role of myeloid cells.</a> Cancer Res. 68, 5501-5504 (2008).</li><li>Liang, J., 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=Neutrophils+promote+the+malignant+glioma+phenotype+through+S100A4.">Neutrophils promote the malignant glioma phenotype through S100A4.</a> Clin Cancer Res Off J Am Assoc Cancer Res. 20, 187-198 (2014).</li><li>Teramukai, S., 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=Pretreatment+neutrophil+count+as+an+independent+prognostic+factor+in+advanced+non-small-cell+lung+cancer:+an+analysis+of+Japan+Multinational+Trial+Organisation+LC00-03.">Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00-03.</a> Eur. 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?...

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><tbody><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco Invitrogen</td><td>21875-034</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td><td>Gibco Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td><td>Life technologies</td><td>25030-024</td><td></td></tr><tr><td>Penicillin streptomycin (Pen Strep)</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Bruton&#039;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&amp;nbsp;</td><td>CliniSciences</td><td>A3001</td><td></td></tr><tr><td>Vincristine&amp;nbsp;</td><td>EG labo</td><td></td><td></td></tr><tr><td>BD Matrigel basement membrane matrix&amp;nbsp;</td><td>BD Biosciences</td><td>354234</td><td>Put at 4 &lt;sup&gt;o&lt;/sup&gt;C overnight (O/N) before the day of the experiment</td></tr><tr><td>Red cell lysis buffer&amp;nbsp;</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Ficoll (Pancoll)</td><td>PAN Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Dextran&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>S3014</td><td></td></tr><tr><td>Annexing V-FLOUS staining kit&amp;nbsp;</td><td>Roche</td><td>11&amp;nbsp;988&amp;nbsp;549&amp;nbsp;001</td><td></td></tr><tr><td>Kit RAL 555 Modified Giemsa staining kit</td><td>Cosmos Biomedical</td><td>CB361550-0000</td><td></td></tr><tr><td>LSRII flow cytometry</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Leica DMR-XA microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cellometer Auto T4 Cell Viability Counter</td><td>Nexcelom&amp;nbsp; Bioscience</td><td></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。 <p class="jove_content" fo:keep-...

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

Universidad de Cartagena UDEC

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

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

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

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

科研

癌症研究

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-基于化验，以监测外核苷酸/核苷代谢的人类慢性淋巴细胞白血病细胞

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

化学

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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 ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.