TB承认囊性纤维化加拿大研究奖学金。

需要注意的是研究伦理委员会（REB）来收集和痰标本从人类受试者的存储。这些研究是由病童医院REB＃1000019444批准。 1.准备CF痰液标本<ol><li>收集患者的痰液标本中的囊性纤维化诊疗常规访问，保持在冰上。 </li><li>在冰上运痰样品收集的第一个小时内，向研究实验室，经过处理。 </li></ol> 2.痰处理<ol><li>记录所得到的痰样品的体积。添加磷酸盐缓冲盐水（PBS）至2倍的样品（ 即，2份PBS中，1份样品）的体积。 </li><li>用移液管混合样品井。涡上1分钟后，完全混合最高设置的样本。 </li><li>等分试样将1ml上述混合物的入适当数量的1.5 ml离心管中并离心以5,000 xg离心在4℃下20分钟。 </li><li>离心后，除去上清液并弃沉淀。 </li><li>过滤通过0.22微米的过滤器消毒上清，在一个干净的离心管收集。 注意:滤液的无菌通过电镀在LB琼脂和接种液体培养基试验。 </li><li>在-80℃，以备将来使用存储痰上清。 注:痰从多个患者也可以下列过滤合并。 </li><li>在使用之前，稀释痰滤液1/10体积/体积中所需的介质（100微升痰，900微升的介质）。 注:在这里，使用标准LB培养基（LB）的媒体。 </li></ol> 3.生物膜形成腔室盖玻片方法<ol><li>在37℃生长在期望的媒体关注隔夜的细菌分离用颤抖的（200转）。 注意:使用了许多不同的细菌，包括临床分离株绿脓杆菌 ， 金黄色葡萄球菌 ，洋葱伯克霍尔德菌复杂和木糖氧化无色杆菌。的媒体选择取决于菌株和感兴趣的条件下，但是LB培养基可以用于最初的实验。 </li><li>从过夜培养，将40微升培养物分成4毫升的新鲜培养基中，并在37℃振荡（200rpm）下在大约0.5 600纳米（OD 600），以获得与光学密度的培养生长3-4小时-0.6。 </li><li>稀释步骤3.2文化在1/5所需的媒体有10％痰滤液或无痰滤液（作为对照）。痰滤液其他浓度可以进行测试（ 即，50％或100％）。 </li><li>用200微升稀释的种子的幻灯片室井。 </li><li>使细菌4小时，在37°C不晃动重视。 </li><li> 4小时后，取出媒体，轻轻地用1X新鲜培养基洗生物膜。用200微升新鲜培养基更换。 注:研究痰的效果上生物膜的FRES^ h媒体应该包含痰上清。 </li><li>允许生物膜到的时间所需量的在37℃不摇动生长，替换媒体每12小时，未经洗涤直到时间为显微镜。 注:研究生物膜药敏痰上清液的效果，抗生素加入到以下生物膜生长的24小时的媒体和被保持在媒体直至染色和生物膜的成像。 </li></ol> 4.染色生物膜和共聚焦显微镜<ol><li>继所需生长时间（24-48小时效果最好），从室移去培养基，并轻轻地用300微升无菌PBS洗涤各室的两倍。 </li><li>通过混合1微升每种染料（在可行性套件中提供）为所需的每个毫升溶液的准备生物膜染色的混合物。使水或媒体解决方案染料。 注:水是由制造商推荐的。 </li><li>加入200μl染料混合物到腔式covergla的每个孔SS和孵育在室温下，在黑暗中45分钟。 </li><li>从腔室中移除染色混合物并用300μl无菌PBS冲洗每个孔中。除去PBS并用清水或媒体取代。 </li><li>通过共聚焦显微镜生物膜的可视化进行。 </li></ol> 5.可视化生物膜与共焦显微镜<ol><li>染色（在1个小时）后，立即阅读室染色生物膜。通过一次染色八月一日至2日孔腔最小化在载玻片的可视化延迟。 </li><li>利用共聚焦显微镜用激光激发和过滤器集收购进行成像。 注:在这里，北极光光谱的激光旋转盘共聚焦系统被用于激励（绿色:561纳米:491nm，红色）。用于发射滤光片套四十零分之五百十五和四十零分之六百二十四的由生物膜生存能力套件形象化的污渍。 </li><li>以使用与相机共聚焦显微镜25X一水的目标图像。 </li><l我&gt;使用Z-堆栈生物膜模型。拍摄图像每隔0.5-1微米从第一焦点对准平面开始到生物膜的最后的聚焦框（通常跨越30-80微米48小时生物膜） </li><li>从每口井以3-5的图像。 注:因此，对于一个8孔腔玻璃罩，将产生24-40图像。 </li><li>保存图像进行分析。 注意:图片应保存为使用COMSTAT 18,19分析OME-TIFF文件。生物膜图像分析说明可以在http://www.comstat.dk/找到。一旦图像被导入，参数，如平均厚度，生物质和表面覆盖率为每个通道（红色和绿色）进行分析。 </li></ol>

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本文描述的方法允许在外源产物的存在下生长的细菌生物膜的可视化。不令人惊讶地，使用这种类型的系统时生产exoproducts的是重要的。例如，二硫苏糖醇（DTT），经常被用来对人痰样品来帮助液化样本。然而，DTT的单独的效果可降低生物膜发展和活力（数据未显示）。因此，对于所有条件适当的控制是必要的。此外，加入的人类痰产品由于这一事实，即每个病人的痰中有一个独特的微生物产生?...

细菌生物膜是微生物组附加到彼此并以自分泌的基质包裹。 1,2-传统上，它们代表细菌物理连接到流量的条件下形成的非生物或生物的表面上。生物膜也已经显示在静态条件（无流动）和远离表面，如在形成于试管热水池或药膜的空气 - 液体界面生长。这些生物膜早已在环境中识别和是主要损害工业过程，因为它们可以在蓄水池或管道形成，导致生物结垢，腐蚀和堵塞。 3,4 生物膜也是在医疗设置关键的，因为它们已被证明参与导管相关感染，囊性纤维化患者的肺感染，以及在许多其他感染。 5,6-一个生物膜感染的标志是德折痕细菌对抗生素的敏感性，并通过先天免疫系统受损的间隙。 7-9最充分研究，涉及基于生物膜感染临床相关情形发生在患者的囊性纤维化（CF），谁是慢性感染绿脓杆菌生物膜。 铜绿假单胞菌能建立慢性感染，使得它很难治疗的过程中发生了一些变化。 10,11生物膜可以激活差异的先天免疫和炎症驱动。 12-14由于这些感染导致CF患者发病率和死亡率，它是要明白，在这方面可以影响生物膜发展的因素是至关重要的。 最近的一项研究表明，宿主因素是在铜绿假单胞菌生物膜聚集体的形成是至关重要的。 15这些生物膜有助于减少对抗生素的敏感性和宿主防御机制。该prese的宿主衍生的因素，如嗜中性粒细胞弹性蛋白酶，以及来自本在CF肺部的微生物分泌的产品NCE，必须大大调制生物膜形成和发展的潜力。 16此外，生物膜与宿主相互作用来调节多种途径表达并开始发炎。而高通量的方法，例如标准的结晶紫测定法中，可以为用户提供关于生物膜过程中的一些信息，响应于这些因素的生物膜的可视化提供更深入的信息。 在这个手稿我们描述了使用来自CF患者的痰因素来研究生物膜的体外发育的方法。此方法允许暴露于使用商业生物膜存活包含痰宿主因素生物膜的快速可视化。这种技术可以用于可视地识别在exogeno存在生物膜生长过程中发生的变化我们的产品，并表示改进的方法来分析各种条件下的生物膜发展的变化。

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

生物膜是由以自分泌的基质包裹细菌的群体。它们在工业污染，以及在许多健康相关性感染的发展和维持中发挥重要作用。一种在人类疾病中最充分描述并研究生物膜发生在囊性纤维化患者的慢性肺部感染。当在主机的情况下学习生物膜，许多因素会影响生物膜的形成和发展。为了鉴定宿主因素如何影响生物膜的形成和发展，我们使用一个静态腔室盖玻片方法中的宿主衍生的因子在痰上清液的形式存在下生长生物膜。细菌接种到商会和暴露于痰滤液。继增长48小时，生物膜沾满共聚焦显微镜和分析之前的商业可行性生物膜套件。以下图像采集，生物膜特性可以使用不同的软件平台来评估。这个方法允许我们想象中不同物质，包括抗生素的存在生物膜生长的关键性质。

实验的整体设计在图1中表示。使用这种协议的提供了一个方便的方法来可视化在生长的不同时间（ 例如，24，48或72小时）的生物膜的变化。重要的是，外源信号，如痰的滤液，可以被添加到可视化生物膜发展的变化。如在图2中看到的那样，10％的痰的滤液的存在可以改变生物膜的结构（ 图2A，下图）。 16这些图像可以...

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

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 ...

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JoVE Journal

Immunology and Infection

8.3K 次观看.  Hospital for Sick Children. 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.

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

科研

免疫与感染

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 ...

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 ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

评估介入疗法对人体粪便微生物群影响的体内批处理培养模型

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

对TNBS介导肠纤维化发展的力学洞察与拉帕霉素抑制作用的评价

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...