Name: a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry
Uploaded: 2013-04-09T13:00:00
Description: Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry Video at JoVE.com

作者要感谢南希·莫拉她的技术援助。 这项工作是由研究资助48573-M和168098从担任Consejo国家CIENCIAŸTECNOLOGIA，墨西哥，的补助IN212308和IN205311转向系总Asuntos DEL个人Academico，墨西哥国立自治大学，墨西哥。

1。从人血中分离中性粒细胞（PMN） <ol><li>约20毫升人体血液中作为抗凝血剂肝素（10 U / ml）的使用。从健康成年志愿者venopuncture采集血液。所有的实验都做生物伦理委员会在研究所德Investigaciones的Biomédicas - 墨西哥国立自治大学的批准下。 </li><li>将2毫升在PBS中的6％葡聚糖T500到15毫升锥形离心管中，并加入10毫升的血液。颠倒试管混合两次或三次，并让它坐45分钟，以允许血沉。 </li><li>在一个新...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+neutrophil+apoptosis:+new+players+enter+the+game.">Regulating neutrophil apoptosis: new players enter the game.</a> Trends Immunol. 32, 117-124 (2011).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+and+NF-&#954;B+in+T+cell+activation:+life+at+the+edge.">Death and NF-&#954;B in T cell activation: life at the edge.</a> Mol. Cell. 11, 551-552 (2003).</li><li>Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., Franzoso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+JNK+signaling+to+NF-&#954;B:+a+key+to+survival.">Linking JNK signaling to NF-&#954;B: a key to survival.</a> J. 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Cancer. 39, 1468-1477 (2003).</li><li>Choi, M., 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=Inhibition+of+NF-&#954;B+by+a+TAT-NEMO-binding+domain+peptide+accelerates+constitutive+apoptosis+and+abrogates+LPS-delayed+neutrophil+apoptosis.">Inhibition of NF-&#954;B by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis.</a> Blood. 102, 2259-2267 (2003).</li><li>Wang, K., 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=Inhibition+of+neutrophil+apoptosis+by+type+1+IFN+depends+on+cross-talk+between+phosphoinositol+3-kinase,+protein+kinase+C-d,+and+NF-&#954;B+signaling+pathways.">Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-d, and NF-&#954;B signaling pathways.</a> J. Immunol. 171, 1035-1041 (2003).</li><li>Schnoor, M., 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=Efficient+non-viral+transfection+of+THP-1+cells.">Efficient non-viral transfection of THP-1 cells.</a> J. Immunol. Meth. 344, 109-115 (2009).</li><li>Garcia-Garcia, E., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+factor+activation+by+Fc&#947;R+in+human+peripheral+blood+neutrophils+detected+by+a+novel+flow+cytometry-based+method.">Nuclear factor activation by Fc&#947;R in human peripheral blood neutrophils detected by a novel flow cytometry-based method.</a> J. Immunol. Meth. 320, 104-118 (2007).</li><li>Garcia-Garcia, E., Nieto-Castaneda, G., Ruiz-Saldana, M., Mora, N., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc&#947;RIIA+and+Fc&#947;RIIIB+mediate+nuclear+factor+activation+through+separate+signaling+pathways+in+human+neuthophils.">Fc&#947;RIIA and Fc&#947;RIIIB mediate nuclear factor activation through separate signaling pathways in human neuthophils.</a> J. Immunol. 182, 4547-4556 (2009).</li></ol>

这里所描述的纯化方法允许隔离未受刺激的嗜中性粒细胞（PMN）的纯度大于95％（通过显微镜观察评估），在很短的时间。有时，中性粒细胞被污染的红细胞，如果后者不完全溶解。通常这并不影响技术，通过流式细胞仪检测，因为红细胞和中性粒细胞可以很容易地被区分为不同的细胞群。隔离PMN然后，可以通过交联的特定受体的特异性单克隆抗体的刺激。事实上，中性粒细胞也可以被刺激的药?...

中性粒细胞是最丰富的外周血中的白细胞1。在炎症和感染过程中嗜中性粒细胞是出现在受影响的部位，在那里他们作为防御2的第一行的第一个细胞。中性粒细胞具有几种抗菌机制的吞噬能力，生产的活性氧的溶解酶脱颗粒，释放和生产，促炎细胞因子4,5。中性粒细胞是短命的细胞得到迅速激活，通过从不同的细胞表面受体的信号。虽然中性粒细胞被认为是终末细胞，由于其短暂的一生，因为它们发生细胞凋亡，除非激活在炎症过程，它现在很清楚，他们也可以修改他们的表型改变特定基因的转录水平。生产的细胞因子5和抑制凋亡7,8两个important活化依赖的细胞功能调节在嗜中性粒细胞中的转录水平。核因子κB（NF-κB）参与细胞因子的生产4，在调节细胞的存活和凋亡9-11在各种细胞类型中的转录控制。 导致核因子激活的信号通路研究报告基因检测或电泳迁移率分析（EMSA）。但是，由于中性粒细胞是短命的细胞，在这些细胞中的转录调控的响应研究不能进行与报告基因检测中，因为不存在有效的中性粒细胞转染技术。 EMSA分析已被用于在嗜中性粒细胞，探讨核因子活化12,13，然而，这种方法是复杂和昂贵的，因为它涉及使用放射性物质。核转染是另一种技术，已被用于成功。LLY转染单核细胞14。因此，至少在理论上，核因子的活化可以在中性粒细胞通过转染检测（尽管效率低）。然而，该技术将更加昂贵，耗时的和可能不太定量。在细胞核中，也可用于免疫染色的细胞的显微分析检测核因素。事实上，我们已检测NF-κB的易位到细胞核中这种方式15。不幸的是，这种技术也是耗时，量变少，和受观察员的偏置。 在这里，我们提出了一个简单而有效的方法，它允许在孤立和immunolabeled的核的流式细胞仪检测和定量的核因子。我们描述技术从人外周血中分离出中性粒细胞，刺激这些细胞通过整合或Fc受体的抗受体抗体，隔离和immunolabel核，并通过流式细胞仪分析核（FIGURE 1）。该方法已成功地用于检测NF-κB和Elk-1的16个核因子中性粒细胞的细胞核。了该方法的灵敏度允许在细胞核中的核因子水平的小的变化的检测。此方法也可用于从其他类型的细胞的核转录因子分析的水平。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

中性粒细胞是最丰富的外周血白细胞。这些细胞是第一次出现在炎症和感染的网站，从而成为第一线的防御入侵的微生物。中性粒细胞具有重要的抗菌功能的吞噬作用，细胞溶解酶的释放，生产的活性氧等。除了这些重要的防御功能，中性粒细胞生产的炎性细胞因子和细胞凋亡的抑制感染，如执行其他任务。细胞因子招募其他的白细胞，帮助清除感染和抑制细胞凋亡，使中性粒细胞在受感染的网站活的时间更长。这些功能是在转录水平的调节。但是，由于中性粒细胞是短命的细胞，在这些细胞中的转录调控的响应研究不能进行与传统的报告基因的方法，因为没有效率cient中性粒细胞转染技术。在这里，我们提出了一个简单而有效的方法，它允许在孤立和immunolabeled的核的流式细胞仪检测和定量的核因子。我们描述的技术来隔离纯粹从人外周血中性粒细胞，刺激这些细胞与抗受体抗体，隔离和immunolabel核，并用流式细胞仪分析核。该方法已成功地用于检测NF-κB和核因子Elk-1的中性粒细胞和其他类型细胞的细胞核中。因此，这种方法代表一个选项用于分析从多种类型的细胞在孤立的核转录因子的激活。

这里所描述的纯化方法通常提供未受刺激的嗜中性粒细胞（PMN）的纯度大于95％（ 图1A）。隔离PMN然后，可以通过交联的特定受体的特异性单克隆抗体的刺激。我们已刺激的PMN通过Fc受体和整合（ 图1B）。一旦受到刺激，中性粒细胞裂解和细胞核被隔离，产量高。细胞核，然后为一个特定的核转录因子核因子κB（NF-κB），与特定的抗体（ 图1B），如immunolabeled?...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry Video at JoVE.com

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry Video (Video) | JoVE

中性粒细胞是白细胞在血液中含量最丰富的。中性粒细胞具有转录调控功能，如生产的炎性细胞因子和抑制细胞凋亡。这些函数可以用这里介绍的方法，它允许核因素，通过流式细胞术检测和定量在孤立的核研究

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

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A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

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Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro Video (Video) | JoVE

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

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

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes Video (Video) | JoVE

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors Video (Video) | JoVE

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus Video (Video) | JoVE

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages Video (Video) | JoVE

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis Video (Video) | JoVE

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...