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>

<ol>
	<li>Cakmak, S., Dales, R. E., Coates, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+air+pollution+increase+the+effect+of+aeroallergens+on+hospitalization+for+asthma?.">Does air pollution increase the effect of aeroallergens on hospitalization for asthma?.</a> J Allergy Clin Immunol. 129 (1), 228-231 (2012).</li><li>Barraza-Villarreal, A., 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=Air+pollution,+airway+inflammation,+and+lung+function+in+a+cohort+study+of+Mexico+City+schoolchildren.">Air pollution, airway inflammation, and lung function in a cohort study of Mexico City schoolchildren.</a> Environ Health Perspect. 116 (6), 832-838 (2008).</li><li>Chen, B. Y., 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+association+of+ambient+air+pollution+with+airway+inflammation+in+schoolchildren.">The association of ambient air pollution with airway inflammation in schoolchildren.</a> Am J Epidemiol. 175 (8), 764-774 (2012).</li><li>Cyprowski, M., Buczynska, A., Szadkowska-Stanczyk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indoor+allergens+in+settled+dust+from+kindergartens+in+city+of+Lodz,+Poland.">Indoor allergens in settled dust from kindergartens in city of Lodz, Poland.</a> Int J Occup Med Environ Health. 26 (6), 890-899 (2013).</li><li>Brough, H. A., 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=Distribution+of+peanut+protein+in+the+home+environment.">Distribution of peanut protein in the home environment.</a> J Allergy Clin Immunol. 132 (3), 623-629 (2013).</li><li>Galli, S. J., Tsai, M., Piliponsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+allergic+inflammation.">The development of allergic inflammation.</a> Nature. 454 (7203), 445-454 (2008).</li><li> World Health Organisation, R. O. f. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenology+and+human+health:+allergic+disorders:+report+on+WHO+meeting+Rome,+Italy.">Phenology and human health: allergic disorders: report on WHO meeting Rome, Italy.</a> , 256 (2003).</li><li>Wuthrich, B., Schindler, C., Leuenberger, P., Ackermann-Liebrich, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+atopy+and+pollinosis+in+the+adult+population+of+Switzerland+(SAPALDIA+study).+Swiss+Study+on+Air+Pollution+and+Lung+Diseases+in+Adults.">Prevalence of atopy and pollinosis in the adult population of Switzerland (SAPALDIA study). Swiss Study on Air Pollution and Lung Diseases in Adults.</a> Int Arch Allergy Immunol. 106 (2), 149-156 (1995).</li><li>Grote, M., Valenta, R., Reichelt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abortive+pollen+germination:+a+mechanism+of+allergen+release+in+birch,+alder,+and+hazel+revealed+by+immunogold+electron+microscopy.">Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy.</a> J Allergy Clin Immunol. 111 (5), 1017-1023 (2003).</li><li>Namork, E., Johansen, B. V., Lovik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+allergens+adsorbed+to+ambient+air+particles+collected+in+four+European+cities.">Detection of allergens adsorbed to ambient air particles collected in four European cities.</a> Toxicol Lett. 165 (1), 71-78 (2006).</li><li>S&#252;ring, 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=PM10+contains+particle-bound+allergens:+Dust+analysis+by+Flow+Cytometry.">PM10 contains particle-bound allergens: Dust analysis by Flow Cytometry.</a> Env Technol Inn. 5, 60-66 (2016).</li><li>Schappi, G. F., Suphioglu, C., Taylor, P. E., Knox, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrations+of+the+major+birch+tree+allergen+Bet+v+1+in+pollen+and+respirable+fine+particles+in+the+atmosphere.">Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere.</a> J Allergy Clin Immunol. 100 (5), 656-661 (1997).</li><li>Buters, J. T., 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+allergen+Bet+v+1+in+fractions+of+ambient+air+deviates+from+birch+pollen+counts.">The allergen Bet v 1 in fractions of ambient air deviates from birch pollen counts.</a> Allergy. 65 (7), 850-858 (2010).</li><li>Buters, J. T. 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=Release+of+Bet+v+1+from+birch+pollen+from+5+European+countries.+Results+from+the+HIALINE+study.">Release of Bet v 1 from birch pollen from 5 European countries. Results from the HIALINE study.</a> Atmos Environ. 55, 496-505 (2012).</li><li>Ormstad, H., Johansen, B. V., Gaarder, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+house+dust+particles+and+diesel+exhaust+particles+as+allergen+carriers.">Airborne house dust particles and diesel exhaust particles as allergen carriers.</a> Clin Exp Allergy. 28 (6), 702-708 (1998).</li><li>Brough, H. A., 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=Peanut+protein+in+household+dust+is+related+to+household+peanut+consumption+and+is+biologically+active.">Peanut protein in household dust is related to household peanut consumption and is biologically active.</a> J Allergy Clin Immunol. 132 (3), 630-638 (2013).</li><li>Jochner, 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=Seasonal+variation+of+birch+and+grass+pollen+loads+and+allergen+release+at+two+sites+in+the+German+Alps.">Seasonal variation of birch and grass pollen loads and allergen release at two sites in the German Alps.</a> Atmos Env. , (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="645">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Teflon filter</td>
			<td style="width:143px;">Pall Life Sciences, USA</td>
			<td style="width:119px;">R2PL047</td>
			<td style="width:167px;">47 mm, 1.0 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">low volume sampler&#160;</td>
			<td>Sven Leckel Ingenieur B&#252;ro GmbH, Germany</td>
			<td style="width:119px;">LVS3</td>
			<td>air flow of 2.3 m3/h</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate-buffered saline</td>
			<td style="width:143px;">Biochrom, Germany</td>
			<td style="width:119px;">L1825</td>
			<td>without Ca/Mg, low endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">electrical toothbrush&#160;</td>
			<td style="width:143px;">Braun, Germany</td>
			<td style="width:119px;">Oral-B Vitality Sensitive</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Casy cell counter&#160;</td>
			<td style="width:143px;">Sch&#228;rfe System GmbH, Germany</td>
			<td style="width:119px;">Model TTC</td>
			<td>range of detectable particle size: 0.7 &#181;m to 45 &#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCanto II&#160;</td>
			<td style="width:143px;">Becton Dickinson, USA</td>
			<td style="width:119px;">3-laser, 8-color (4-2-2)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS Diva Software v6.1.3&#160;</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)&#160;</td>
			<td style="width:143px;">Sigma-Aldrich, USA</td>
			<td style="width:119px;">A2153-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">monoclonal mouse IgG1 antibody against Bet v 1</td>
			<td style="width:143px;">Indoor Biotechnologies, UK</td>
			<td style="width:119px;">&#160;MA-3B4</td>
			<td style="width:167px;">clone MA-3B4</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">APC (Allophycocyanin)-labeled secondary anti-Mouse IgG1 antibody&#160;</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">560089</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SPSSTM software version 18&#160;</td>
			<td style="width:143px;">PASW Statistics 18, Hongkong, China</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri Dish</td>
			<td style="width:143px;">Gosselin, France</td>
			<td style="width:119px;">BP50-02</td>
			<td>D 55mm, H 15mm&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS Tube&#160;</td>
			<td style="width:143px;">Becton Dickinson, USA</td>
			<td style="width:119px;">REF 352054</td>
			<td>5ml Polystyrene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASYton</td>
			<td style="width:143px;">Roche 
			Germany</td>
			<td style="width:119px;">REF 05651808001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Matrix Blank Tubes</td>
			<td style="width:143px;">Thermo Scientific, USA</td>
			<td align="right" style="width:119px;">4140</td>
			<td>1,4 ml, PP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge</td>
			<td style="width:143px;">Heraeus, Thermo Scientific</td>
			<td style="width:119px;">Megafuge 40R</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vacuum Pump</td>
			<td style="width:143px;">INTEGRA Biosciences AG, Switzerland</td>
			<td style="width:119px;">Model 158 320</td>
			<td>Inetrgra Vacusafe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">recombinant Bet v 1a antigen&#160;</td>
			<td style="width:143px;">Indoor Biotechnologies, UK</td>
			<td style="width:119px;">LTR-BV1A-1</td>
			<td>Concentration:&#160; 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

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

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

科研

免疫与感染

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

压力处理下细菌生长的多尺度分析

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...