LMW和RHK的缅因大学的研究生院生物医学科学和工程（GSBSE）支持; RHK还得到了缅因州的农业和林业试验场。提供额外拨款，由国家普通医学科学研究所（NIH P20-GM103423），缅因州的农业和林业试验场（批准号ME08004-10，JAG），大学缅因州ADVANCE:涨潮中心（NSF格兰特＃1008498）和研究入门格兰特（JAG）从PhRMA的基础药理/毒理。我们感谢博士。的大卫Holowka和芭芭拉·贝尔德抗原和细胞。雏哈什米，亚历杭德罗·贝莱斯，和安德鲁Abovian的设备和订单的帮助，我们非常感谢。这是缅因州农业和林业试验场出版3311。

的NaAl 等[1]在2004年，开发高通量检测脱颗粒肥大细胞生物传感器。它是一个强大的检测，我们已经适应了我们的TCS研究，在这部影片中详述。在此之前的NaAl 等。1法，肥大细胞脱颗粒已常规通过评估β-氨基己糖苷酶的59-61，但这些早期的方法，利用一次读取一个样本的荧光。重要的是，的NaAl 等 。他们利用高通量的方法样品一读，一时间在荧光酶标仪和以前方法之间?...

肥大细胞高度免疫效应细胞作为主要介质哮喘，过敏，寄生虫国防和癌变13-16粒。他们居住在几乎所有的血管组织15，他们在那里安全地存储在细胞质颗粒，直到过敏和炎症介质激活脱颗粒。脱颗粒胞吐作用的膜结合的颗粒，这会导致释放的药理学活性的介质如组胺，类胰蛋白酶和白三烯15。这个过程的结果我的启动类型是至关重要安装防御寄生虫以及发起过敏，哮喘和致癌反应15过敏反应。 肥大细胞和嗜碱性粒细胞表达FcεRI受体，高亲和力受体，免疫球蛋白E（IgE）的17。暴露于变应原或抗原导致聚集多个绑定的IgE的FcεRI受体17，它是这个S邻-被称为"交联"的IgE结合的Fc受体发起的脱颗粒过程:酪氨酸磷酸化事件，激活磷脂酶C，钙外流，从内部存储，进入细胞内的钙流入18级联。此钙离子内流所需的脱颗粒，并进一步与膜之前，使颗粒胞吐15信号颗粒融合。在实验中，钙离子载体可以直接穿过细胞膜19，它本质上绕过所有的信号转导的钙离子内流步骤20步骤之前，用于穿梭钙，允许通路目标识别为上游或下游有毒物钙信号20。 的脱颗粒可以迅速和有效地测量通过监测组胺6一起从线性的颗粒，释放到细胞上清液中β-氨基己糖苷酶的释放，但我更加容易去检测使用一个简单的酶 - 底物反应，酶标仪测定荧光产物。这微孔板检测，详载于协议部分，是基于一个健壮的方法最初开发的NaAl 等。1，量化的荧光底物4 -甲基-N-乙酰-β-D-葡萄糖苷的裂解β-氨基己糖苷酶。我们已经修改了药物和毒物检测，以测试效果，三氯生在这里强调。此方法可靠地量化脱颗粒，是一种廉价的替代，例如，流式细胞仪为基础的检测方法（21），有可能很好地适合各种各样的抗过敏的药物的高通量筛选，以及免疫系统的或过敏的化学物质。根据2007年美国国家研究委员会的报告"在21 世纪的毒性测试，最后这一点是特别重要的:愿景和STRAT埃及"（ <a href="http://www.nap.edu/openbook.php?record_id=11970">http://www.nap.edu/openbook.php?record_id=11970</a> ）的毒理学试验，利用高通量细胞培养，以减少昂贵的使用传统的实验动物，如小鼠的发展，主张。脱粒协议的NaAl 等制定和修改由我们2，利用RBL-2H3细胞系，这是一个公认的模型对人体黏膜肥大细胞或嗜碱性粒细胞3-5同源。 （RBL-2H3细胞培养的方法在哈钦森等人详述22）。本试剂盒也有可能适应任何附加的肥大细胞类型。 三氯生（TCS）是一种广谱抗菌药物，已经使用了30年以上，在医院，个人护理产品和消费品23,24。行动模式为TCS的抗菌特性是抑制脂肪酸的合成，有可能通过抑制烯酰 - 酰基载体蛋白还原酶25,26。它被发现在全球范围广泛的消费性产品如沐浴露，洗手液，牙膏，漱口水，洗手液，其浓度为0.3％或10 mM的24。 TCS的广泛使用，导致在人类27-29河流和溪流30的检测水平。由Allmyr 等人所做的一项研究27表明，TCS和它的代谢产物是存在于血浆和哺乳期的母亲的牛奶。重要的是，TCS很容易被吸收到皮肤31-37。 Queckenberg 等37〜12小时内进入人体皮肤吸收10％〜70毫米TCS霜，造成重大浓度在皮肤上，肥大细胞的所在。 TCS已经临床管理人力过敏性皮肤病7-11，但一直默默无闻的机制，TCS缓解过敏性皮肤病38。使用荧光酶标仪测定的详细资料D在这段视频中，我们最近表明，在浓度低至2微米，TCS，极大地挫伤了肥大细胞功能和脱颗粒，提供了一个可能的解释这些临床数据2。除了 ​​这些临床数据提供了一个解释，我们的研究结果帕尔默等人。2表明，TCS目标的下游信号分子钙离子内流。由于钙信号在许多免疫学和其他生物过程的重要性，TCS上的各种必要的生物过程可能会产生不利的影响。事实上，Udoji 等39表明TCS抑制人体自然杀伤细胞溶解活性，另一个重要的先天免疫功能。 除了 ​​其潜在的过敏性皮肤病（或反过来说，作为一个immunotoxicant）作为一种辅助治疗帮助，TCS也可能是一种内分泌干扰物40-49。因此，如何准备这种化学物质在溶液中，我明确的程序s的兴趣毒理学。因为TCS是一个小的疏水性分子，通常用于有机载体，使其更溶于水。在已经过测试，其中TCS的毒性研究，制备涉及在水中溶解的有机溶剂，如乙醇，丙酮，或石油2,50,51与援助。然而，很多时候这些溶剂具有生物活性，从 ​​而解释复杂的化学测试数据51。事实上，根据到Rufli 等。52和53，这是建议的测试解决方案，为水生生物毒性实验准备用物理方法，化学方法，由于化学溶剂创建毒性文物的潜在。我们先前已经表明，TCS溶解在0.24％的乙醇/水（体积/体积），超声处理30分钟挫伤RBL肥大细胞脱颗粒2。浓度高于0.24％的乙醇溶液，在已被证明能抑制肥大细胞的退化会使nulation 54,55例有机溶剂毒性研究的潜在混杂因素的影响。 它不仅是重要的考虑溶剂对生物体或细胞用于研究的效果，而且还监测效果的溶剂测试化学品本身是很重要的。例如，Skaare的等51发现:TCS，溶解在聚乙二醇（通常在牙膏和漱口水）减弱健康女性女性的抗细菌和抗牙斑的作用，而在油中的溶解引起的功能完全丧失。因此，不同溶剂的能力来调节毒物和药物，包括TCS，效果应该考虑在试验设计。油或有香味的添加剂的使用，可能会干扰的影响，在各种产品中的TCS 50,51。 在努力以消除需要使用有机溶剂，我们改进我们的方法溶解TCS 2时，通过消除使用的有机溶胶发泄。在本协议中，我们直接TCS颗粒溶解成水缓冲热（≤50℃），然后验证的浓度这TCS股票的紫外 - 可见分光光度法。这些改进是可能的，因为TCS是易溶于水高达40μM（ <a href="http://www.epa.gov/oppsrrd1/REDs/2340red.pdf">http://www.epa.gov/oppsrrd1/REDs/2340red.pdf</a> ），并已被证明至抗蚀剂的降解，当加热到50℃（ <a href="http://oehha.ca.gov/prop65/public_meetings/052909coms/triclosan/ciba3.pdf">呻/ oehha.ca.gov/prop65/public_meetings/052909coms/triclosan/ciba3.pdf</a> ）56,57。我们也有额外的好处，紫外-可见分光光度法，TCS也被称为摩尔消光系数为4200 L / mol /平方厘米的12强烈地吸收280 nm处58。 到一个缓冲区中的有机溶剂不借助溶解TCS颗粒，该协议提供了一个简单而有效的方法，包括低成本和快速验证浓度，并描述了一个强大的荧光微孔板检测监控化学作用于肥大细胞脱颗粒。

a microplate assay to assess chemical effects on rbl-2h3 mast cell degranulation: effects of triclosan without use of an organic solvent

肥大细胞在过敏性疾病和寄生虫的免疫防御中扮演着重要的角色。一旦被激活（ 例如通过过敏原），他们脱颗粒，一个过程，结果在胞吐过敏介质。调制肥大细胞脱颗粒，药物和毒物，可能对人体健康有正面或负面的影响。肥大细胞功能已被详细解剖大鼠嗜碱性白血病肥大细胞（RBL-2H3），使用一种被广泛接受的模型，人类粘膜肥大细胞3-5。肥大细胞颗粒成分和过敏介质β-氨基己糖苷酶的释放，这是线性地随着组胺从肥大细胞6，可以容易且可靠地进行测量通过荧光底物的反应，得到衡量的荧光强度在酶标仪测定是服从高通量研究1。最初发表的NaAl 等1，我们已经适应了这个脱颗粒检测的筛选f药物和毒物，并在这里展示其使用。 三氯生是一种广谱抗菌剂，是存在于许多消费产品，并已发现在人的皮肤过敏性疾病的7-11是一种治疗援助，尽管这种效果的机理是未知的。在这里，我们展示了一个检测三氯生对肥大细胞脱颗粒的效果。我们最近发现，三氯生的强烈影响肥大细胞功能2。在努力避免使用有机溶剂，三氯生与热和搅拌下直接进入含水缓冲液溶解，所得浓度经使用紫外-可见分光光度法（ε280 = 4200 L / M /厘米）12。此协议有可能被使用的各种化学品，以确定其对肥大细胞脱颗粒，并更广泛地说，他们的过敏性潜力。

当加热至50℃下进行90分钟的UV-Vis吸收光谱为TCS产生一个强大的，平滑的曲线之间的260〜300nm的，在280nm处具有峰值，如在图1中示出。紫外-可见分光光度法，因此，一个重要的工具，可以用来计算浓度，既然发布的在280nm处的摩尔吸光系数为4200升/ 12摩尔/厘米。我们已经发现，TCS不会掉出的时间范围内的整个脱颗粒实验的溶液后，50℃加热（数据未示出）。 使用这种加热方法直接进入含?...

Watch this Scientific Journal Video about A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent at JoVE.com

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

肥大细胞脱颗粒，释放过敏介质，过敏，哮喘和寄生虫防御是很重要的。在这里，我们展示了对脱颗粒的药物和毒物的影响评估的技术 ，方法，最近利用抗菌剂三氯生2，表现出强大的抑制作用。

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒:三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

a-microplate-assay-to-assess-chemical-effects-on-rbl-2h3-mast-cell

Research

JoVE Journal

Immunology and Infection

39.9K 次观看.  University of Maine, Orono. 肥大细胞脱颗粒，释放过敏介质，过敏，哮喘和寄生虫防御是很重要的。在这里，我们展示了对脱颗粒的药物和毒物的影响评估的技术 ，方法，最近利用抗菌剂三氯生2，表现出强大的抑制作用。 

视频: A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

测量抗体乳鼠心肌细胞对细胞功能的影响

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

科研

免疫与感染

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

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

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 diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

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 ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒:三氯生的影响不使用有机溶剂的化学效应

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