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>在一个新的15毫升锥形离心管中，将5毫升的Ficoll-Paque联合应用。 </li><li>以富含白细胞的血浆上方形成沉淀的红细胞，并仔细吸取试剂的Ficoll-Paque联合形成的第二层的顶部上。确保有两个阶段。 </li><li>在4℃下离心20分钟，在516×g离心离心分离后，在血浆和Ficoll-Paque的相间有单核细胞的层。氖在试管底部utrophils是。 </li><li>消除上清液，通过轻敲管针对机架打破细胞沉淀，重悬细胞，加入10毫升冷的（4℃）的PBS。 注：重悬细胞通过上下吹打不推荐使用，因为这是太破坏细胞。 </li><li>细胞悬浮液转移到50毫升锥形离心管中，离心，在290×g离心5分钟，在4℃下</li><li>如前额的细胞沉淀，并加入10毫升冷的（4℃）低渗溶液（0.2％的NaCl，1％BSA的20mM的Hepes，pH值= 7.4），以溶解红细胞。混合管用手轻轻摇晃了整整一分钟。 </li><li>加入10毫升冷的（4℃）高渗溶液（1.6％的NaCl，1％BSA的20mM HEPES，pH值= 7.4），混合，并计数细胞用血细胞计数器（通常&gt; 95％是中性粒细胞）。 注意：保持冰的细胞计数管的细胞悬液。 </li><li>像以前那样离心，重悬在10 PMN7细胞/毫升在低温（4℃）PBS。请在冰上。 注：嗜中性粒细胞仍然可行的和功能性的，在4℃下进行约6〜8小时。 </li></ol> 2。激活中性粒细胞<ol><li>将100μl的PMN悬浮液（1×10 7个细胞/ ml）至1.5 ml Eppendorf管中。 注：样品管，应该至少一式两份。通常阴性对照应该包括：第一的抗体只，和二次抗体。 </li><li>添加相应的抗integrin或抗Fc受体的单克隆抗体（mAb），10微克/毫升，并在冰上孵育15分钟。 </li><li>通过加入1毫升的冷（4℃）PBS中，于1,743 xg离心（4,500 rpm转速）在离心的离心，吸取上清液。洗净PMN的</li><li>额攻丝针对机架的底部的管，将细胞沉淀，加入1ml冷的PBS中，洗2次以上，在前面的步骤。 注意：这些洗涤除去未结合的抗体。 </li><li>重悬相同（PMNitial）量的温水（37°C）PBS含60μg/ ml的F（AB&#39;）2的羊抗鼠IgG。 注：的二次抗小鼠IgG抗体将结合到第一抗受体抗体，将导致该受体的交联。 </li><li>在37℃下孵育1至20分钟，这取决于对核因子的权益。 NF-κB的通常为15分钟，和Elk-1的通常为5分钟。 注：孵化的细胞在37℃下诱导受体交联，这是不可能发生的，在4℃下</li><li>加入1ml冷PBS，并在1743 XG中的微量离心3分钟。 </li><li>除去上清液</li><li>冻结PMN立即在dry-ice/ethanol浴，让他们有10分钟。 </li></ol> 3。隔离和固定的核<ol><li>冷冻中性粒细胞沉淀重悬在100μl冷（4℃）的低渗缓冲液（10mM的HEPES，10 mM KCl中，1.5毫摩尔MgCl 2，和 1 mM的新鲜增值DL-二硫苏糖醇（DTT），pH值= 7.9）。建议采取管满分在dry-ice/ethanol浴一步法由一个，并加入低渗缓冲液之前，擦拭干净的试管底部。 </li><li>置于冰上，并检查核完整的细胞核悬浮液用台盼蓝染色的等分。核举目四顾，蓝色。完整的中性粒细胞被染色，并显示为蓝色颗粒形状不规则的细胞碎片。 注：如果细胞核悬浮液中含有许多完整的细胞或碎片，最好是放弃的准备和与其它样本重复上述步骤。 </li><li>离心机核在775 XG（3000转）的微量离心10分钟，寒冷的房间内。在这一点上核是很脆弱的，格外小心，应采取冷的样品，而不是在过大的力离心。 </li><li>除去上清液非常小心的移液。 </li><li>加入100μl冷的4％多聚甲醛固定核。 注：颗粒是非常松散的D添加缓冲液是足够的重悬细胞核。上下吹打应加以避免。 </li><li>在冰上孵育20分钟。 </li><li> immunolabel原子核用于流式细胞仪，或将其维持长达24小时，在4℃下</li></ol> 4。核应用免疫流式细胞仪分析<ol><li>离心1743的XG（4500转），原子核的离心1分钟，然后取出上清液很温柔的移液。 </li><li>加入100μl的冷（4℃），0.1％的Triton X-100，在PBS中的4％多聚甲醛透性核。在冰中孵育10分钟。 </li><li>在1743 XG中的微量离心机透核，小心地取出上清液。此时细胞核粒料不附加在试管底部。建议再次离心，沉淀得到宽松。 </li><li>重悬细胞核在500μl的冷的4％牛胎儿血清（FBS）在PBS中，以阻断非特异性结合位点，并在冰上孵育20分钟。离心分离原子核在1743 XG的离心3分钟，小心地取出上清液。 </li><li>重悬在100μl冷PBS含4％FBS和2.5微克/毫升的单克隆抗体对核因子的利息的原子核。在冰上孵育20分钟。 注：常住阴性对照只应包括抗核因子抗体，FITC标记的二次抗体只。 </li><li>用500μl冷的PBS洗两次，用4％的FBS和在1743×g离心3分钟离心。 </li><li>非常小心除去上清液，重悬核在冷PBS100μl的含有4％FBS和10微克/毫升，相应的FITC-标记的第二抗体，并在冰上孵育20分钟。 </li><li>用500μl的冷PBS洗净细胞核两次与4％的FBS，如在前面的步骤。 </li><li>重悬在400μl的冷的4％多聚甲醛的PBS中的原子核。 注：核被放置在多聚甲醛，让样品数天而不会受到污染，被存储血清的存在下，在可能发生的问题。 </li><li>分析立即用流式细胞仪或存放在黑暗中，在4°C长达三天。 </li></ol> 5。流式细胞仪分析<ol><li>分析immunolabeled核流式细胞仪在FACScan（Becton Dickinson公司，新泽西州富兰克林湖）或类似的设备。 </li><li>调整收购的设置，在日志规模在10-1：FSC，SSC在日志规模在196和栅极核点图。 </li><li>采集每个样品10000核。 </li><li>通过分析FITC染色的细胞核的荧光FL-1通道（506 nm）的日志规模在400。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

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 at JoVE.com

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

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

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

a-simple-efficient-method-to-detect-nuclear-factor-activation-human

Research

JoVE Journal

Immunology and Infection

17.7K 次观看.  University of Alberta. 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

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

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

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

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

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

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

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

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

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

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

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 ...