The authors would like to thank Katrin Bossmann, Anett Neumann and Eike Wolter (German Environment Agency) for their valuable preparatory work.

注:本协议描述了单克隆抗体PM10颗粒对Bet v 1的，主要的桦树花粉抗原成分，再加上别藻蓝蛋白（APC）间接染色（单克隆小鼠IgG1抗体，克隆MA-3B4）标记的二次抗体（抗-mouse IgG1抗体，克隆A85-1）和流式细胞随后的分析。适当的其他抗体可用，此方法也可能延伸到检测结合到周围空气中的颗粒的其他抗原。 1. PM10采样<ol><li>从聚四氟乙烯（PTFE）的环境空气收集PM10滤波器使用低容量采样2.3 立方米 /小时（ 图1）的流量。用于这里所描述的实验中取样的表征在16中找到。运行时间取决于所需的可吸入颗粒物的量（通常为1至10天）。 </li><li>在温育时间结束时，从采样移除过滤器，并在将其冻结-20℃下直到使用。 </li></ol><img alt="图1" src="/files/ftp_upload/54721/54721fig1.jpg" /> 图1.低容积PM10采样器。一个低容积PM10采样的例子。 <a href="http://ecsource.jove.com/files/ftp_upload/54721/54721fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.去除可吸入颗粒物和粒子计数<ol><li>让约5分钟的PTFE过滤解冻。然后，将过滤器在一个干净的聚苯乙烯培养皿（ 图2A）。取一个新的培养皿的每个滤波器中，如果一个以上的过滤器进行处理。 </li><li>接着，覆盖用磷酸盐缓冲盐水（PBS）的PTFE过滤器。此协议，建立了每ml 8x10的6颗粒的最终PM10浓度（参见步骤3.3）。以获得至少该浓度，使用PBS中的下列经验体积与覆盖在过滤器:如果PM10收集时间为&lt;<html> 2天后，使用2个毫升对于孵育时间≥2天，采用4毫升 注意:为了增加PM10悬浮液的粒子浓度，从不同的过滤器混悬液可被汇集，如果这是合适的。 </li><li>握住镊子和刷PTFE过滤器与一个敏感刷头1分钟（ 图2B，2C）的电牙刷。传送颗粒的PBS悬浮，以后称为PM10混悬液，向一干净反应管。 </li></ol><img alt="图2" src="/files/ftp_upload/54721/54721fig2.jpg" /> 图2. PM10除去有电牙刷与采样PM10聚四氟乙烯过滤器被放置在一个聚苯乙烯培养皿（A）和叠置用4ml PBS中。然后，PM10与一个电牙刷去除（A:刷牙1分钟后:刷牙和C之前）。="http://ecsource.jove.com/files/ftp_upload/54721/54721fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <ol start="4"><li>测量PM10颗粒， 例如浓度，通过使用粒子计数器。稀PM10悬浮例如50微升在10ml等渗测量缓冲器的充足体积，测量三次，并计算每毫升微粒的平均总数。一定要使用它可以检测相关的大小颗粒的粒子计数器。 </li></ol> 3. Bet v 1的染色<ol><li>计算样品的分析所需的反应管的数量:至少有三个需要反应管:（i）一种管只有样品（天然对照）（ii）一种管与样品加二级抗体（阴性对照），和（ iii）一名管品加一级和二级抗体（具体的样品）。如果是第一次测量，准备（i）至少两个反应管（ii）中，（三）有足够的材料来适当调整设置流式细胞仪（见步骤4.1）。 </li><li>计算PM10悬浮于反应管的数量所需的量。每个反应管需要50微升悬浮液。 </li><li>调整在步骤2.4悬浮液中加入PBS中适当量的在步骤3.2计算每毫升8x10的6颗粒的终浓度的量测得的粒子浓度。 注:其余PM10悬浮液可在-20℃用于以后的实验被冻结，尽管该协议新鲜建议制备PM10悬浮液。 </li><li>块通过在0.02％的最终浓度，补充PM10悬浮液用牛血清白蛋白（BSA）使用的储备溶液用PBS制备如1％BSA的非特异性结合。短暂涡旋并孵育在室温下20分钟。 </li><li>用于向通过流式细胞术来分析各样品，转移50微升悬浮液从步骤3.4到交流精益反应管中。添加抗Bet v 1的单克隆小鼠抗体以0.02微克/微升到指定用于特定染色，涡旋简要反应管的终浓度和孵育在室温下60分钟。 <ol><li>让与指定为本地控制和负控制也留在室温下60分钟的PM10-BSA悬浮反应管。 </li></ol></li><li>通过加入500微升的PBS补充有0.02％BSA的每个反应管中，涡旋短暂离心样品随后4700 xg离心在室温下5分钟洗净所有样本。通过使用真空泵小心弃上清。 </li><li>重复步骤3.6。 </li><li>确定APC标记的第二抗小鼠IgG1抗体的所需的总量:溶解在50μlPBS中补充有每反应管0.02％BSA的1微克抗体。稀释抗体的适当量与补充有0.02％BSA的PBS的各卷。 </li><li>加入50μl稀释的第二抗体的所有反应管中，除了指定本地控制的反应管中。补充后者用50μl的PBS补充有0.02％牛血清白蛋白。涡旋所有样品几秒钟。 </li><li>孵育所有样品在黑暗中30分钟。 </li><li>重复步骤3.6的两倍。 </li><li>加入50μl的PBS到每个反应管中，涡旋和流式细胞仪分析上的流动的样品。 </li></ol> 4.流式细胞仪分析<ol><li>使用本机的控制，调整以下流式细胞仪参数来优化数据分析。 <ol><li>通过使用设备控制板上显示的前向散射（FSC）阈值控制，设置FSC阈值的最低值（200），并开始分析。 </li><li>通过使用分散电压控制器，以这种方式调节FSC和侧向散射（SSC），所有的可吸入颗粒物的颗粒可以被检测和该颗粒群大致位于第在SSC轴的下半部分中的FSC轴以及e中间。 </li><li>通过使用荧光电压控制器，调节APC电压和另一个荧光电压像异硫氰酸荧光素（FITC），它不象在APC同一波长，如有必要，不发出。确保，即所有的粒子在两种荧光轴的下半部分中可见。 </li></ol></li><li>连续，检查每个样本包括本机控制和FSC，SSC，APC和FITC数据存储每个样品至少10,000颗粒。 </li><li>评价与相应的软件的数据。具体样品中的吸附变应原含量可以用两种方式来量化: <ol><li>分析所有粒子（中值），为Bet v 1的负载超过所有PM10颗粒的量度的APC荧光强度。 注意:如果该特定样品含有变应原，APC荧光强度应在特定样品中的增加相比，阴性对照。与此相反，其它氟escence强度（ 例如 ，FITC荧光强度）应不显著变化。 </li><li>计算PM10颗粒的结合的抗Bet v 1的抗体的百分比。从而，在阴性对照中，设置围绕考虑APC阳性颗粒的栅极，复制和这个门粘贴到特定样品和减去的APC阳性的粒子的比例在来自APC正粒子的具体样品中的百分比的阴性对照。 </li></ol></li></ol>

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The authors have nothing to disclose.

该协议的一个关键步骤是使用用于从环境空气中的PM10颗粒的收集适当的过滤器（见步骤1.1）。该过滤器必须足够强以承受与电牙刷刷牙，并且不是所有的过滤材料满足这一要求。染色协议建立与每毫升8x10的6颗粒的PM10颗粒浓度。但是，如果该材料是有限的，样品池是不恰当的，则该方法可能会起到很好，但抗体浓度（参见步骤3.5和3.8）可能必须进行调整。 PM10颗粒的Bet v 1的染色不导致正和负染粒子不同的种群。这可能是由不同量的Bet v 1的变应原吸附在每个从非常小到一个高量的颗粒造成的。这可能导致在APC信号的膨胀从而转移人口朝APC阳性。因为很难对正从负颗粒分离，使用两个量化的方法来确定在PM10&gt; 0.5试样的Bet v 1的内容的差异:通过测量所有粒子的中值的APC荧光强度（ⅰ）的相对定量和（ii）测定APC的阳性颗粒的百分比。关于从低和高花粉季节PM10&gt; 0.5的颗粒的Bet v 1的负荷，这两种方法显示类似的结果。静止，建议由所有粒子的中值荧光强度相对定量，因为它是独立放置的栅极，因此可能不容易出错。 迄今为止许多研究审查通过提取相应的过敏原和随后的定量与ELISA 5,12-14,17环境空气颗粒物过敏原含量。有此处描述的过程和quantificatio之间的根本区别Ñ采用ELISA:酶联免疫吸附量化所提取的并溶解抗原，而流式细胞分析颗粒结合的抗原。通过ELISA测试PM10样品的Bet v 1的负荷（N = 8）低于1.2纳克/毫升的检测限（未示出数据）。同样，Buters和其他标识在PM &lt;2.5μm的级分，并在10微米仅约7％&gt; PM&gt;2.5μm的部分，但在PM环境空气13&gt; 10微米级分在93％以上没有Bet v 1的。 ELISA法的一方面和另一方面的FACS分析的对比结果，可通过结合不同的灵敏度的检测方法的差异造成的。进一步的研究但需要充分了解这种差异。 一种方法，以可视化粒子结合抗原扫描电子显微镜10,14。用扫描电子显微镜，Ormstad 等悬浮颗粒磨砂的表面上的可视化Bet v 1的- [R烟尘颗粒采样在高花粉季节，并就在低花粉季节15取样颗粒程度较轻。此外，发现从花粉，乳胶和还β葡聚糖过敏原被吸附到燃烧颗粒在环境空气10。该方法中，然而，不允许这些结合颗粒的过敏原的定量。 通过使用流式细胞仪，粒子结合Bet v 1的过敏原可以量化。因此，流式细胞仪可以提供表征PM10的10〜0.5μm的生物组分与手头其他合适的抗体的新方法，这种方法可能会扩展到对周围环境空气中的颗粒， 如霉菌，尘螨检测其他抗原过敏原或LPS。作为PM10颗粒很容易吸附不仅生物材料，而且化学品和金属，非特异性抗体的结合可以，但是，构成问题。如果一个新的抗体测试，一个关键的步骤是证明具体绑定ING。这可以这样做，例如，具有染色11之前相应抗原阻断特异性抗体的结合的能力。 由于Bet v 1的含量周围空气可以从桦树花粉数12,13,18不同，过敏症状或许可以与相关过敏原水平比花粉更好计数14,18。因此，与临床数据结合所提出的方法，能够在以后的实验中，研究过敏反应桦木是否对应于PM10&gt; 0.5的Bet v 1的变应原的负荷。

空气污染被认为是增加了近几十年来观察1-3呼吸道过敏的发生率和严重程度的一个重要的环保事业。此外，在常见的过敏原的灰尘4,5-分布越来越大的兴趣。 桦树花粉可以激起花粉症但也可以是过敏性哮喘6-8的一个重要触发。整个桦树花粉是不可能进入下呼吸道或PM10被发现作为其大小（直径22微米）的结果。然而，桦树花粉过敏样Bet v 1的，主要的桦树花粉过敏原成分，可以花粉破裂后9被释放，并可以绑定到周围空气中的颗粒10，从而可能进入下呼吸道，。事实上，已经表明，可吸入颗粒物可以含有具有生物活性的变应原作为来自花粉过敏先证者11表明由嗜碱细胞的体外活化</suP&gt;。 PM10样品中的Bet v 1的变应原含量进行了研究，通过提取的各变应原和随后的量化采用ELISA 12-14。用ELISA技术，溶解变应原进行了测定，但应原吸附颗粒的量仍然不明。扫描电子显微镜观察发现过敏原吸附颗粒，但并没有让量化10,15。 本研究采用流式细胞仪量化Bet v 1的加载PM10颗粒周围的空气样本中的比例。由于流动的检测极限仪只颗粒大于0.5μm，可以检查。 PM10的&gt; 0.5微米的部分将进一步被称为PM10&gt; 0.5。

<table><tbody><tr><td>Teflon filter</td><td>Pall Life Sciences, USA</td><td>R2PL047</td><td>47 mm, 1.0 &amp;micro;m</td></tr><tr><td>low volume sampler</td><td>Sven Leckel Ingenieur B&amp;uuml;ro GmbH, Germany</td><td>LVS3</td><td>air flow of 2.3 m&lt;sup&gt;3&lt;/sup&gt;/hr</td></tr><tr><td>Phosphate-buffered saline</td><td>Biochrom, Germany</td><td>L1825</td><td>without Ca/Mg, low endotoxin</td></tr><tr><td>electrical toothbrush&amp;nbsp;</td><td>Braun, Germany</td><td>Oral-B Vitality Sensitive</td><td></td></tr><tr><td>Casy cell counter&amp;nbsp;</td><td>Sch&amp;auml;rfe System GmbH, Germany</td><td>Model TTC</td><td>range of detectable particle size: 0.7 &amp;micro;m to 45&amp;nbsp;&amp;micro;m</td></tr><tr><td>FACSCanto II&amp;nbsp;</td><td>Becton Dickinson, USA</td><td>3-laser, 8-color (4-2-2)</td><td></td></tr><tr><td>FACS Diva Software v6.1.3&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>bovine serum albumin (BSA)&amp;nbsp;</td><td>Sigma-Aldrich, USA</td><td>A2153-10G</td><td></td></tr><tr><td>monoclonal mouse IgG1 antibody against Bet v 1</td><td>Indoor Biotechnologies, UK</td><td>&amp;nbsp;MA-3B4</td><td>clone MA-3B4</td></tr><tr><td>APC (Allophycocyanin)-labeled secondary anti-Mouse IgG1 antibody&amp;nbsp;</td><td>Becton Dickinson</td><td>560089</td><td>clone A85-1</td></tr><tr><td>SPSSTM software version 18&amp;nbsp;</td><td>PASW Statistics 18, Hongkong, China</td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Gosselin, France</td><td>BP50-02</td><td>D 55&amp;nbsp;mm, H 15&amp;nbsp;mm</td></tr><tr><td>FACS Tube&amp;nbsp;</td><td>Becton Dickinson, USA</td><td>REF 352054</td><td>5 ml Polystyrene</td></tr><tr><td>CASYton</td><td>Roche&lt;br/&gt; Germany</td><td>REF 05651808001</td><td></td></tr><tr><td>Matrix Blank Tubes</td><td>Thermo Scientific, USA</td><td>4140</td><td>1.4 ml, PP</td></tr><tr><td>Centrifuge</td><td>Heraeus, Thermo Scientific</td><td>Megafuge 40R</td><td></td></tr><tr><td>Vacuum Pump</td><td>INTEGRA Biosciences AG, Switzerland</td><td>Model 158 320</td><td>Inetrgra Vacusafe</td></tr><tr><td>recombinant Bet v 1a antigen&amp;nbsp;</td><td>Indoor Biotechnologies, UK</td><td>LTR-BV1A-1</td><td>Concentration: 2.0 mg/ml</td></tr></tbody></table>

flow cytometric analysis of particle-bound bet v 1 allergen in pm10

流式细胞术是一种广泛用于量化的悬浮固体，如在从0.5至几十直径微米的尺寸范围的细胞或细菌的方法。除了前向和侧向散射性质的表征，它使得能够使用荧光标记的标记物的样抗体，以检测相应结构。使用间接抗体染色，流式细胞仪在这里采用来量化桦树花粉过敏原（准确地说Bet v 1的）-loaded的0.5至10微米的颗粒直径可吸入颗粒物（PM10，粒度≤10μm的直径）。作为吸附变应原的载体可能他们运送到下呼吸道，在那里他们可以引发过敏反应PM10颗粒可以采取行动。 到目前为止PM10的过敏原内容已经研究了酶联免疫吸附测定（ELISA）和扫描电子显微镜的手段。 ELISA措施的解除，在颗粒-bound过敏原。相比扫描电子显微镜，它可以可视化变应原吸附颗粒，流式细胞仪可另外量化。环境空气中过敏原的含量可从白桦花粉计数偏离，过敏症状或许可以与相关过敏原接触比花粉量更好。在临床资料的同时，该方法提供了机会，在今后的实验来检验过敏反应是否桦树花粉抗原与PM10颗粒&gt; 0.5微米的Bet v 1的过敏原含量有关。

Bet v 1的变应原吸附到PM10&gt; 0.5的颗粒通过间接抗体染色和随后的分析上的流式细胞仪定量。从高花粉季节PM10一个样品充当模板。如在步骤3.1中所述，阴性对照由用APC孵育PM10颗粒标记的第二抗体只（ 图3A）。用抗Bet v 1的抗体加二抗染色的PM10颗粒显示的过敏原装载颗粒（ 图3B）。如步骤4.3中所述，使用量化的变应原负载的两种方式:在一方面，被分析的所有颗粒的APC荧光强度的中值是137的阴性对照和904的具体示例。另一方面，具有结合的抗Bet v 1的抗体的粒子的比例被确定:一个闸门在阴性对照周围设置在APC阳性颗粒并随后共花衣服并粘贴到特定的样本。在阴性对照中，可吸入颗粒物&gt; 0.5的颗粒的3％被认为APC阳性。假阳性的颗粒的此百分比是从正面粒子的具体样品中的百分比从而导致77.5％的APC阳性PM10&gt;特定样品中的0.5颗粒减去。为了证明所观察到的抗赌注结合V 1抗体是特异性的，结合能力被阻止与染色前的相应的抗原。这减少了69％的抗Bet v 1的抗体的结合，如由APC的荧光强度定量，84％，如果由Bet v 1的正面的PM10&gt; 0.5的颗粒（ 图3C）的百分比定量。 <img alt="图3" src="/files/ftp_upload/54721/54721fig3.jpg" /> 图3.粒子结合Bet v 1的过敏原可以通过从高埗一PM10样品的流式细胞仪。APC荧光强度进行可视化LLEN季节仅与APC染色标记的第二抗体（A）中，与抗Bet v 1的主要抗体，随后与在APC染色标记的第二抗体（B）和阻断用重组Bet v 1的抗原的第一抗体后（℃ ）。该波门P + APC围绕认为APC积极颗粒设置（显示为红色）和百分比分别给出。这个数字已经从11略有修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54721/54721fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> 为了验证这种方法是否可以采用，以揭示的高和低花粉季节PM10&gt; 0.5样品的吸附Bet v 1的内容量的差异，从最高的13 PM10样品和从低花粉季节6 PM10样品进行了分析。 图4描绘了在APC荧光强度和在Bet v 1的正面的PM10&gt;从高花粉季节相比从低花粉季节PM10样品PM10样品0.5颗粒的比例显著差异。这两种量化方法在此表现出类似的结果。 <img alt="图4" src="/files/ftp_upload/54721/54721fig4.jpg" /> 图4.低和高花粉季节PM10样品中的吸附投注诉量差异1.低花粉季节PM10秋/冬2013取样（N = 6）2012年5月和2013年的高花粉季节PM10（N = 13）。 （A）PM10的APC荧光强度&gt;高花粉季节0.5颗粒比低花粉季节（平均/最大/最小高花粉季节显著较高:796/313/1097;平均/最大/最小低花粉季节:197 / 85/277）中。 （B）PM10高花粉季节包含significan比PM10更TLY Bet v 1的阳性PM10&gt; 0.5的颗粒从低花粉季节（平均/最大/最小高花粉季节:45.2 / 18.5 / 74.5;平均/最大/最小低花粉季节:11.8 / 4.4 / 19.8）。箱形图显示中位值（框的内侧线），25和75百分位数，分别为（盒的下和上边界）以及最小和最大值（须）。 *** P &lt;0.001与低花粉季节，曼惠特尼U检验。这个数字已经从11略有修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54721/54721fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10 at JoVE.com

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

在这里，我们提出了一个协议，通过流式细胞仪量化过敏原吸附颗粒。周围的颗粒物质的颗粒可充当吸附变应原的载体。我们在这里显示，流式细胞仪中，一种方法被广泛用于表征悬浮固体&gt;在直径为0.5微米，可以用来测量这些过敏原载粒子。

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

flow-cytometric-analysis-of-particle-bound-bet-v-1-allergen-in-pm10

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.5K 次观看.  Federal Environment Agency. 在这里，我们提出了一个协议，通过流式细胞仪量化过敏原吸附颗粒。周围的颗粒物质的颗粒可充当吸附变应原的载体。我们在这里显示，流式细胞仪中，一种方法被广泛用于表征悬浮固体>在直径为0.5微米，可以用来测量这些过敏原载粒子。 

视频: Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

科研

生物工程

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

免疫与感染

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

不同环境条件下生物气溶胶的组成及分布分析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

环境科学

Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) from two different study sites in a resource-constrained setting of Tamil Nadu, India. Particulate matter PM2.5 (particles smaller than 2.5 microns in aerodynamic diameter), carbon monoxide (CO), and black carbon (BC) were measured in pregnant mothers (M), other adult women (OAW), and children (C) at various times over a 4 year period. In addition, stove usage monitoring (SUMs) with data-logging thermometers and ambient measurements of air pollution were carried out. Furthermore, the feasibility of collecting biological samples (urine and dried blood spots [DBSs]) from study participants at the field sites was successfully demonstrated. Based on findings from this and earlier studies, the methods used here have enhanced the data quality and avoided issues with household air pollution and biological sample collection in resource-constrained situations. The procedures established may be a valuable educational tool and resource for researchers conducting similar air pollution and health studies in India and other low- and middle-income countries (LMICs).

We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

可视化印度家庭空气污染干预网络试验的暴露和生物标志物评估的现场数据收集程序

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) ...

<meta charset="utf8"></head><body>通过诱导痰液分析定量肺巨噬细胞中的碳

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

<meta charset="utf8"></head><body>测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

中国南方与过敏性鼻炎和/或哮喘的花粉过敏原研究的成分分析诊断方法

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

医学

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

粒子结合Bet v 1的变应原PM10的流式细胞分析

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测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

中国南方与过敏性鼻炎和/或哮喘的花粉过敏原研究的成分分析诊断方法

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎