作者要感谢为这项研究提供资金的囊性纤维化基金会和MedImmune慷慨捐赠的抗Psl0096抗体。在这项研究中，成像是在多伦多大学的CAMiLoD成像设施进行的。

收集和储存人类受试者的痰液样本需要获得研究伦理委员会 （REB） 的批准。本文介绍的研究已获得病童医院 REB#1000058579 的批准。
1. 痰液收集
<ol><li>将咳出的痰液储存在无菌收集杯中，并在固定前立即在4°C下储存最多24小时。 注意:痰液在4°C下放置时间过长而不固定会导致细胞降解，特别是白细胞降解。最好尽快固定。</li>
	<li>将痰液样本转移到无菌的 15 mL 管中。</li>
	<li>向痰液样本中加入等体积的 4% 多聚甲醛 （PFA）。例如，如果痰液样本为 0.5 mL，则添加 0.5 mL 的 4% PFA。通过轻轻倒置混合。</li>
	<li>将痰液在4°C孵育过夜。</li>
	<li>通过向每 2 mL 固定痰液中加入 5 mL 磷酸盐缓冲盐水 （PBS） 来洗涤样品。 注意:此洗涤步骤不需要离心。</li>
	<li>用移液管小心地除去上清液。 注意: 避免用移液器吸痰，将尖端远离痰液，然后非常缓慢地吸出表面液体。洗涤步骤不涉及离心，以免干扰痰塞的结构完整性。</li>
	<li>再重复步骤 1.6-1.7 两次。</li>
	<li>将沉淀重悬于含有0.01%（w / v）叠氮化钠的PBS体积的2倍中。</li>
	<li>将痰液储存在4°C。</li>
</ol>2. MiPACT痰液的处理
<ol><li>将水凝胶组分（30%29:1丙烯酰胺:双丙烯酰胺，硬化剂和PBS）在含有厌氧包装的密封容器中脱气72小时。 注意:厌氧包装和密封容器是必要的，因为氧气会抑制丙烯酰胺聚合。或者，可以通过在厌氧罩中执行此步骤、对密封容器施加真空或通过丙烯酰胺混合物鼓泡 N2 气体来去除氧气。<ol><li>将 2 mL 的 30% 29:1 丙烯酰胺:双丙烯酰胺溶液加入 15 mL 试管中，在密封的厌氧容器内取下盖子。</li>
		<li>在 15 mL 试管中向 5 mL PBS 中加入 0.5 g 固化剂，制成 10% （w/v） 固化剂的浓缩原液。将盖子从管子上取下，存放在密封的厌氧容器中。</li>
		<li>将几毫升无菌 PBS 留在厌氧容器内的试管中，盖上盖子。</li>
	</ol></li>
	<li>在 15 mL 试管中制备 5 mL 水凝胶溶液，终浓度为 0.2% 固化剂和 4% 29:1 丙烯酰胺:双丙烯酰胺的 PBS 溶液。倒置混合并过滤灭菌。</li>
	<li>从冰箱中取出痰液样本，然后在无菌条件下用手术刀切成小段（直径约5毫米）。 注意:如果痰液非常流动，那么仍然可以执行此步骤，但是，在使用手术刀分离所需部分之前，需要增加耐心和练习，用镊子从储存溶液中取出痰液。</li>
	<li>将切割的痰液样品放入8室盖玻片的孔中。</li>
	<li>将步骤2.2中的300μL过滤灭菌水凝胶溶液加入到每个含有痰液的孔中。</li>
	<li>将8室盖玻片置于装有厌氧包的密封容器内，在37°C下放置3小时。 注:一旦聚合，嵌入痰液的水凝胶应为坚硬凝胶的稠度。</li>
	<li>将固化的水凝胶痰液样品转移到含有5mL的8%十二烷基硫酸钠（SDS），pH 8的15mL培养管中，并让样品在37°C下清除3-14天（有或没有摇动），直到痰液变得透明。 注意:清除时间取决于痰液成分，通常可以通过摇晃来减少。但是，请注意不要将振荡速度提高到破坏样品完整性的程度。与富含粘液的样品相比，富含 DNA 的样品需要更长的时间才能清除。</li>
	<li>将 8% SDS 溶液倒入废物收集容器中。使用无菌镊子将每个水凝胶包埋的样品转移到无菌 50 mL 锥形管中。向50mL锥形管中的每个管中加入10mL PBS以洗涤水凝胶，并在倾析前让溶液静置30分钟至1小时。重复此操作 2 次以上。 注意:此洗涤步骤不涉及离心。用移液管小心除去多余的液体和上清液。</li>
	<li>将洗涤的样品储存在含有0.01%（w / v）叠氮化钠和1x RNase抑制剂的PBS中，温度为4°C。</li>
</ol>3. 水凝胶荧光 原 位杂交（FISH）方案
<ol><li>使用无菌镊子从其储存溶液中取出水凝胶样品，并将样品放在无菌表面（如载玻片或培养皿）上。</li>
	<li>使用无菌手术刀将水凝胶切成~1毫米厚的切片。</li>
	<li>将 1 mm 水凝胶切片放入无菌 1.5 mL 管内。</li>
	<li>在 1.5 mL 试管中制备 1 mL 杂交缓冲液（25% 甲酰胺、0.9 M NaCl、20 mM Tris-HCl [pH 7.6]、0.01% SDS、纯化和去离子化 H2O）。</li>
	<li>将荧光标记的PseaerA探针（150.7nM12）加入杂交缓冲液中，并通过倒置混合。 注意:福尔马林溶液应远离明火。杂交缓冲液可以预先制备，并在-20°C13下以等分试样储存。</li>
	<li>向每个~1mm水凝胶切片中加入200-500μL杂交缓冲液，并确保整个水凝胶样品浸没。</li>
	<li>通过将PseaerA探针置于黑暗中~18-24小时，在46°C下不摇晃，使PseaerA探针与水凝胶样品杂交。</li>
	<li>将杂交缓冲液倒入废物收集容器中。</li>
	<li>通过向每个 1.5 mL 试管中加入 1 mL 洗涤缓冲液，然后将其取出，用过滤灭菌的洗涤缓冲液（337.5 mM NaCl、20 mM Tris-HCl、5 mM EDTA（乙二胺四乙酸）[pH 7.2]、0.01% SDS 和纯化和去离子的 H2O）冲洗样品一次。 注意:洗涤缓冲液可以提前制备并在室温 （RT） 下储存。</li>
	<li>向试管中加入1mL新鲜洗涤缓冲液，然后将样品在48°C下在黑暗中孵育6小时，不要摇晃。</li>
</ol>4. 水凝胶与Psl0096抗体结合
<ol><li>使用 1 mL 移液器无菌除去洗涤缓冲液。</li>
	<li>通过加入和除去 1 mL 的 2% BSA 溶液，用 PBS 溶液中的 2% BSA （w/v） 冲洗样品。</li>
	<li>向水凝胶样品中加入 500 μL 2% BSA/PBS 溶液以阻断非特异性蛋白结合。然后将样品在黑暗中在室温下孵育过夜，不要摇晃。</li>
	<li>使用 1 mL 移液管无菌除去封闭溶液。</li>
	<li>通过在 500 μL 新鲜的 2% BSA/PBS 中将抗体稀释至 0.112 μg/mL 的终浓度来制备 Psl0096-Texas Red 抗体溶液。</li>
	<li>将 500 μL 抗体溶液加入水凝胶样品中，并在室温下孵育 6 小时，避光，不要摇晃。</li>
</ol>5. DAPI（4′，6′-二脒基-2-苯基吲哚）染色
<ol><li>通过将 40 g 非离子密度梯度培养基、30 μL 吐温 20、3 μg 叠氮化钠和 30 mL PBS 加入到含有磁力搅拌棒的烧瓶中来制备折射率匹配溶液 （RIMS）。在磁力搅拌器上搅拌溶液15分钟，或直至完全溶解。</li>
	<li>过滤 使用 10 mL 注射器和无菌 0.2 μm 过滤器将溶液灭菌到 50 mL 锥形管中。 注意:溶液可以在4°C下储存数月。</li>
	<li>用无菌 1 mL 移液管取出 Psl0096-Texas Red 抗体溶液。</li>
	<li>通过加入然后除去 1 mL PBS 冲洗水凝胶样品。</li>
	<li>将水凝胶样品与 250 μL RIMS 溶液和 10 μg/mL DAPI 在室温下在室温下孵育，并在黑暗中轻轻摇动过夜。</li>
	<li>在进行共聚焦成像之前，将样品安装到0.9 mm或1.7 mm灌注室上，并用玻璃盖玻片密封。 注:在 FISH 和/或免疫组化之后以及浸入 RIMS 之前，如果需要痰粘液的可视化，可以应用荧光凝集素染色12。</li>
</ol>6. 影像学检查
<ol><li>使用标准技术以 25 倍、40 倍、63 倍或 100 倍放大倍率进行共聚焦激光扫描显微镜成像。</li>
</ol>

<ol>
	<li>Banerjee, D., Stableforth, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+treatment+of+respiratory+pseudomonas+infection+in+cystic+fibrosis:+What+drug+and+which+way.">The treatment of respiratory pseudomonas infection in cystic fibrosis: What drug and which way.</a> Drugs. 60 (5), 1053-1064 (2000).</li><li>Rosenfeld, M., Ramsey, B. W., Gibson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+acquisition+in+young+patients+with+cystic+fibrosis:+Pathophysiology,+diagnosis,+and+management.">Pseudomonas acquisition in young patients with cystic fibrosis: Pathophysiology, diagnosis, and management.</a> Current Opinion in Pulmonary Medicine. 9 (6), 492-497 (2003).</li><li>Ciofu, O., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+Resistance+of+Pseudomonas+aeruginosa+Biofilms+to+Antimicrobial+Agents-How+P.+aeruginosa+Can+Escape+Antibiotics.">Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics.</a> Frontiers in Microbiology. 10, 913 (2019).</li><li>Billings, N., 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+Extracellular+Matrix+Component+Psl+Provides+Fast-Acting+Antibiotic+Defense+in+Pseudomonas+aeruginosa+Biofilms.">The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms.</a> PLoS Pathogens. 9 (8), 1003526 (2013).</li><li>Franklin, M. J., Nivens, D. E., Weadge, J. T., Lynne Howell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+the+Pseudomonas+aeruginosa+extracellular+polysaccharides,+alginate,+Pel,+and+Psl.">Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl.</a> Frontiers in Microbiology. 2, 167 (2011).</li><li>Yang, L., 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=Polysaccharides+serve+as+scaffold+of+biofilms+formed+by+mucoid+Pseudomonas+aeruginosa.">Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa.</a> FEMS Immunology and Medical Microbiology. 65 (2), 366-376 (2012).</li><li>Wozniak, D. J., 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=Alginate+is+not+a+significant+component+of+the+extracellular+polysaccharide+matrix+of+PA14+and+PAO1+Pseudomonas+aeruginosa+biofilms.">Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7907-7912 (2003).</li><li>Baker, P., 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=Exopolysaccharide+biosynthetic+glycoside+hydrolases+can+be+utilized+to+disrupt+and+prevent+Pseudomonas+aeruginosa+biofilms.">Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.</a> Science Advances. 2 (5), 1-9 (2016).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Chung, 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li><li>Treweek, J. B., 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=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nature Protocols. 10 (11), 1860-1896 (2015).</li><li>DePas, W. H., 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=Exposing+the+three-dimensional+biogeography+and+metabolic+states+of+pathogens+in+cystic+fibrosis+sputum+via+hydrogel+embedding,+clearing,+and+rRNA+labeling.">Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling.</a> mBio. 7 (5), 1-11 (2016).</li><li>Murray, M. P., Doherty, C. J., Govan, J. R. W., Hill, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+processing+time+and+storage+of+sputum+influence+quantitative+bacteriology+in+bronchiectasis.">Do processing time and storage of sputum influence quantitative bacteriology in bronchiectasis.</a> Journal of Medical Microbiology. 59 (7), 829-833 (2010).</li><li>Efthimiadis, A., Jayaram, L., Weston, S., Carruthers, S., Hargreave, 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=Induced+sputum:+Time+from+expectoration+to+processing.">Induced sputum: Time from expectoration to processing.</a> European Respiratory Journal. 19 (4), 706-708 (2002).</li><li>Chao, Y., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fixation+methods+for+observation+of+bacterial+cell+morphology+and+surface+ultrastructures+by+atomic+force+microscopy.">Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy.</a> Applied Microbiology and Biotechnology. 92 (2), 381-392 (2011).</li><li>St-Laurent, J., Boulay, M. E., Prince, P., Bissonnette, E., Boulet, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell+fixation+methods+of+induced+sputum+specimens:+An+immunocytochemical+analysis.">Comparison of cell fixation methods of induced sputum specimens: An immunocytochemical analysis.</a> Journal of Immunological Methods. 308 (1-2), 36-42 (2006).</li><li>Hogardt, 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=Specific+and+rapid+detection+by+fluorescent+situ+hybridization+of+bacteria+in+clinical+samples+obtained+from+cystic+fibrosis+patients.">Specific and rapid detection by fluorescent situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 38 (2), 818-825 (2000).</li><li>Hoffmann, N., 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=Erratum:+Novel+mouse+model+of+chronic+Pseudomonas+aeruginosa+lung+infection+mimicking+cystic+fibrosis.">Erratum: Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis.</a> Infection and Immunity. 73 (8), 5290 (2005).</li><li>Nair, B., 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=Utility+of+Gram+staining+for+evaluation+of+the+quality+of+cystic+fibrosis+sputum+samples.">Utility of Gram staining for evaluation of the quality of cystic fibrosis sputum samples.</a> Journal of Clinical Microbiology. 40 (8), 2791-2794 (2002).</li><li>Howlin, R. P., 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=Low-Dose+Nitric+Oxide+as+Targeted+Anti-biofilm+Adjunctive+Therapy+to+Treat+Chronic+Pseudomonas+aeruginosa+Infection+in+Cystic+Fibrosis.">Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis.</a> Molecular Therapy. 25 (9), 2104-2116 (2017).</li><li>Pestrak, M. J., 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=Treatment+with+the+Pseudomonas+aeruginosa+glycoside+hydrolase+PslG+combats+wound+infection+by+improving+antibiotic+efficacy+and+host+innate+immune+activity.">Treatment with the Pseudomonas aeruginosa glycoside hydrolase PslG combats wound infection by improving antibiotic efficacy and host innate immune activity.</a> Antimicrobial Agents and Chemotherapy. 63 (6), 00234 (2019).</li></ol>

该协议的目的是了解CF患者痰液中 铜绿假单胞菌 细胞的原位组织。痰液样本应储存在4°C，如果不能立即固定，则应进行处理。已经证明，如果在4°C下储存1小时，24小时或48小时处理，痰中的 铜绿假单胞菌 细胞数量不会显着变化，但如果在25°C下放置24或48小时，细菌细胞计数将由于细菌生长而显着增加14。对于这项研究，痰液样本在4°C下储存，吐痰后最多24小时。?...

在这项研究中，设计了实验来可视化小儿囊性纤维化 （CF） 患者痰液中铜绿假单胞菌的体内结构。铜绿假单胞菌感染在 30-40% 的儿科 CF 人群中发展为慢性;慢性感染一旦形成，几乎不可能消除1.来自早期感染患者的铜绿假单胞菌分离株通常更易受到抗菌药物的影响，因此，这些药物用抗假单胞菌抗生素治疗，以防止慢性感染的建立2。不幸的是，并非所有铜绿假单胞菌分离株在抗生素治疗后都能从肺部有效清除。与抗生素失败相关的确切机制尚未完全阐明。先前的研究表明，生物膜细胞密度、聚集和多糖产生的变化会影响抗生素疗效3。铜绿假单胞菌产生三种细胞外多糖:Pel、Psl 和海藻酸盐4。大多数铜绿假单胞菌菌株具有表达每种胞外多糖的遗传能力，尽管通常一种多糖主要表达5.海藻酸外多糖与CF肺的慢性感染有关，导致粘液样表型6,7。多糖 Pel 和 Psl 具有多种功能，包括帮助初始附着和维持生物膜结构，以及赋予抗生素耐药性8。
已经为各种样品类型开发了旨在可视化组织体内结构的方法9,10,11。最近，它们被定制用于可视化 CF 患者痰液中的体内微生物群落12。DePas等人，2016年12开发了专门用于鉴定痰中微生物群落的组织清除方案的优化。术语 MiPACT 代表 Passive CLARITY technique 之后的 microbial i识别，用于清除 CF 痰液11,12。对于组织透明化技术，首先将标本固定，然后使其透明，同时保持其固有结构完好无损，以便进行染色和显微观察11。固定和清除CF痰液样本使研究人员能够回答与生物膜结构、细菌细胞密度、多种微生物关联以及病原体与宿主细胞之间的关联相关的问题。直接检查保存在痰液中的细菌的优点是可以在宿主特定的环境中对其进行分析和可视化。尽管在实验室中用于实验的临床分离株的体外生长可以提供非常丰富的信息，但这些方法无法完全重建CF肺环境，导致实验室结果与患者结果脱节。
这里介绍的方法可用于固定和清除痰液以可视化细菌，无论是来自 CF 患者还是患有其他呼吸道感染的患者。本文描述的染色和显微分析的具体类型是荧光 原位 杂交（FISH），然后在水凝胶内进行抗Psl抗体结合，然后通过共聚焦激光扫描显微镜（CLSM）进行分析。组织清除后，也可以应用其他免疫组化和显微镜方法。

<table>
	<tbody>
		<tr>
			<td>29:1 acrylamide bisacrylamide, 30 % solution</td>
			<td>BioRad</td>
			<td>161-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Chambered Coverglass Nunc Lab-Tek</td>
			<td>ThermoFischer Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerogen2.5L</td>
			<td>Oxid Inc.</td>
			<td>35108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwell perfusion chambers</td>
			<td>Electron Microscopry Sciences</td>
			<td>70326 -12/-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoDenz</td>
			<td>Sigma</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protect RNA Rnase Inhibitor</td>
			<td>Sigma</td>
			<td>R7387</td>
			<td></td>
		</tr>
		<tr>
			<td>PseaerA - GGTAACCGTCCCCCTTGC</td>
			<td>Eurofins</td>
			<td></td>
			<td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td>
		</tr>
		<tr>
			<td>Psl0096-Texas Red</td>
			<td>Medimmune</td>
			<td></td>
			<td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td>
		</tr>
		<tr>
			<td>VA-044 Hardener</td>
			<td>Wako</td>
			<td>27776-21-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of pseudomonas aeruginosa within the sputum of cystic fibrosis patients

及早发现和根除囊性纤维化患者肺部的铜绿假单胞菌可以减少发生慢性感染的机会。慢性铜绿假单胞菌感染的发展与肺功能下降和发病率增加有关。因此，对阐明抗生素治疗未能根除铜绿假单胞菌的原因非常感兴趣，铜绿假单胞菌发生在大约 10-40% 的儿科患者中。可能影响宿主对铜绿假单胞菌清除和抗生素敏感性的众多因素之一是空间组织（如聚集或生物膜形成）和多糖产生的变化。因此，我们有兴趣可视化CF患者痰液中铜绿假单胞菌的原位特征。将痰液样品包埋到水凝胶基质中后，将组织透明化技术应用于痰液样品，以保留相对于宿主细胞的 3D 结构。组织清除后，添加荧光标记物和染料以允许可视化。荧光原位杂交用于细菌细胞的可视化，荧光标记的抗Psl抗体的结合用于胞外多糖的可视化和DAPI染色对宿主细胞的染色以获得结构见解。这些方法允许通过共聚焦激光扫描显微镜对CF患者痰中的铜绿假单胞菌进行高分辨率成像。

实验的整体设计总结在图 1 和 图2中。 图 1 总结了痰液处理和痰液清除方案。痰液处理和清除可能需要长达 17 天的时间。但是，该方案可以停止，并且可以在用PFA固定（第2天）或组织清除后（第5-17天，取决于清除时间）后储存样品。 在图2中，总结了FISH和抗体结合方案。FISH 和抗体染色方案需要 4 天才能?...

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

该方案提供了囊性纤维化患者痰液中细菌细胞和多糖合成位点（Psl）多糖的可视化方法。

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

visualization-pseudomonas-aeruginosa-within-sputum-cystic-fibrosis

Research

JoVE Journal

Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

2.9K 次观看.  Hospital for Sick Children. 该方案提供了囊性纤维化患者痰液中细菌细胞和多糖合成位点（Psl）多糖的可视化方法。

视频: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

囊性纤维化患者痰液中铜绿假单胞菌的可视化

摘要

探索更多视频

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

铜绿假单胞菌和酿酒酵母流细胞生物膜

RNA的分离绿脓杆菌殖民小鼠胃肠道

使用人工痰介质测试抗生素药效试验

长期慢性

绿脓杆菌引起的肺损伤模型

IL-8瞬时转化的小鼠模型

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

摘要

探索更多视频

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

铜绿假单胞菌和酿酒酵母流细胞生物膜

RNA的分离绿脓杆菌殖民小鼠胃肠道

使用人工痰介质测试抗生素药效试验

长期慢性

绿脓杆菌引起的肺损伤模型

IL-8瞬时转化的小鼠模型

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

囊性纤维化患者痰液中铜绿假单胞菌的可视化