Name: quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software
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Les auteurs tiennent à remercier la Fondation de la fibrose kystique d’avoir financé cette recherche.

1. Collecte d’isolats bactériens
<ol><li>Obtenir des isolats de P. aeruginosa d’une cohorte de patients pédiatriques atteints de fibrose kystique subissant un traitement d’éradication par tobramycine inhalée à l’Hôpital SickKids (Toronto). Congeler les isolats à -80 °C dans du citrate de glycérol et une sous-culture au moins trois fois avant de les utiliser.</li>
</ol>2. Formation du biofilm in vitro
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<ol>
	<li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nature Reviews Microbiology. 8, 623-633 (2010).</li><li>Flemming, H. C., 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=Biofilms:+an+emergent+form+of+bacterial+life.">Biofilms: an emergent form of bacterial life.</a> Nature Reviews Microbiology. 14, 563-575 (2016).</li><li>Rybtke, M., Hultqvist, L. 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A., Khaddour, G., Fran&#231;ois, B., Massart, T. 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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=Objective+threshold+selection+procedure+(OTS)+for+segmentation+of+scanning+laser+confocal+microscope+images.">Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images.</a> Journal of Microbiological Methods. 47, 169-180 (2001).</li><li>Arena, E. 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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=Alginate+production+affects+Pseudomonas+aeruginosa+biofilm+development+and+architecture,+but+is+not+essential+for+biofilm+formation.">Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.</a> Journal of Medical Microbiology. 53, 679-690 (2004).</li><li>Reichhardt, C., Parsek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+scanning+microscopy+for+analysis+of+Pseudomonas+aeruginosa+biofilm+architecture+and+matrix+localization.">Confocal laser scanning microscopy for analysis of Pseudomonas aeruginosa biofilm architecture and matrix localization.</a> Frontiers in Microbiology. 10, 677 (2019).</li><li>Yang, X., Beyenal, H., Harkin, G., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+biofilm+structure+using+image+analysis.">Quantifying biofilm structure using image analysis.</a> Journal of Microbiological Methods. 39, 109-119 (2000).</li><li>Yang, X., Beyenal, H., Harkin, G., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biofilm+image+thresholding+methods.">Evaluation of biofilm image thresholding methods.</a> Water Research. 35, 1149-1158 (2001).</li><li>Ross, S. 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F., 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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426, 306-310 (2003).</li><li>Srinandan, C. S., Jadav, V., Cecilia, D., Nerurkar, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrients+determine+the+spatial+architecture+of+Paracoccus+sp.+biofilm.">Nutrients determine the spatial architecture of Paracoccus sp. biofilm.</a> Biofouling. 26, 449-459 (2010).</li><li>Ramos, I., Dietrich, L. E., Price-Whelan, A., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenazines+affect+biofilm+formation+by+Pseudomonas+aeruginosa+in+similar+ways+at+various+scales.">Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales.</a> Research in Microbiology. 161, 187-191 (2010).</li><li>Ma, L., Jackson, K. D., Landry, R. M., Parsek, M. R., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Pseudomonas+aeruginosa+conditional+psl+variants+reveals+roles+for+the+psl+polysaccharide+in+adhesion+and+maintaining+biofilm+structure+postattachment.">Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment.</a> Journal of Bacteriology. 188, 8213-8221 (2006).</li></ol>

Il n’existe pas de méthode prescrite pour comparer quantitativement les images tridimensionnelles des structures de biofilms in vitro , et les procédures décrites dans ce contexte sont souvent difficiles à normaliser en raison de la variabilité inter-opérateurs20. Ainsi, ce protocole offre un cadre simple et reproductible pour les applications COMSTAT cherchant à quantifier les changements dans l’architecture du biofilm in vitro dans diverses conditions antimicrobienne...

Les biofilms sont des agrégats de micro-organismes orientés dans une matrice de substances polymères extracellulaires (EPS) autoproduites. La matrice EPS est très complexe, composée principalement de cellules bactériennes, d’eau, de protéines, de polysaccharides, de lipides et d’acides nucléiques1, qui rendent tous les biofilms nettement différents des cellules planctoniques libres. Les biofilms EPS sont adhérents les uns aux autres et à diverses surfaces. La matrice EPS possède des propriétés qui interviennent dans l’échange de métabolites, de matériel génétique et de composés utilisés pour la signalisation et la défense intercellulaires2. Ces propriétés fournissent collectivement aux biofilms une intégrité structurelle et une protection contre les facteurs de stress externes, contribuant à l’évasion immunitaire et à la résistance aux antimicrobiens3.
Pseudomonas aeruginosa est un pathogène nosocomial bien connu, connu pour adopter une stratégie de croissance évasive du biofilm en réponse aux antimicrobiens. Un excellent exemple de cela se produit chez les patients atteints de la maladie génétique récessive, la mucoviscidose (FK). Les biofilms jouent un rôle central dans le développement de P. aeruginosa4 résistant aux antimicrobiens et permettent l’établissement d’une infection pulmonaire chronique chez les patients atteints de mucoviscidose, entraînant un déclin accéléré de la fonction pulmonaire et une mortalité prématurée5. Par conséquent, des études in vitro sur biofilm sont réalisées pour tester l’efficacité des antibiotiques et de nouveaux agents anti-infectieux contre les isolats de P. aeruginosa obtenus chez des patients atteints de FC 6,7. Après la formation du biofilm, les antimicrobiens sont appliqués à l’extérieur de la structure, et la microscopie confocale à balayage laser (CLSM) est utilisée pour générer des reconstructions tridimensionnelles à haute résolution des segments de biofilm. Il est courant d’utiliser ensuite le logiciel informatique, COMSTAT, en tant que plug-in à ImageJ, pour quantifier les changements dans l’architecture du biofilm 8,9,10,11.
Bien que COMSTAT soit utile pour quantifier la structure du biofilm, la reproductibilité et la standardisation de l’analyse d’images sont moins souvent abordées. Par exemple, la procédure de traitement d’image, effectuée avant l’exécution de COMSTAT, est objective, mais contient un élément de subjectivité lors de la définition des seuils d’image12,13. De la même manière, le programme COMSTAT permet à l’opérateur de choisir des conditions et des paramètres de base à avancés pour la segmentation d’images ainsi que dix fonctions d’analyse quantitative (par exemple, la distribution de l’épaisseur, la surface, la biomasse, le coefficient de rugosité sans dimension). La multitude d’options d’utilisation, combinée aux différents niveaux d’expertise de l’opérateur, peut entraîner des rapports erronés sur le comportement du biofilm.
Ainsi, l’objectif de ce protocole est de présenter une méthode relativement simple pour la comparaison quantitative des structures de biofilms in vitro à l’aide de COMSTAT. Ici, des images tridimensionnelles de segments de biofilm d’un isolat de P. aeruginosa de la FC sont capturées par CLSM à l’aide du modèle14 à chambre de protection, une technique établie utilisée pour réaliser des expériences in vitro reproductibles sur biofilm. Utilisant COMSTAT comme plug-in d’ImageJ, cette méthode permet aux chercheurs d’identifier quantitativement les changements dans l’architecture du biofilm en présence d’antimicrobiens dans diverses conditions. Dans l’ensemble, cette méthode vise à éliminer les variations subjectives associées au fonctionnement manuel de COMSTAT, facilitant ainsi la standardisation des protocoles entre les centres.

<table>
	<tbody>
		<tr>
			<td>Anti-Psl mAb, Psl0096</td>
			<td>Medimmune</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar (TSA with 5 % Sheep Blood) Medium</td>
			<td>Fisher Scientific</td>
			<td>R01200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eight-well Chambered Coverglass w/ non-removable wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen SYTO 9 Green Fluorescent Nucleic Acid Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S34854</td>
			<td></td>
		</tr>
		<tr>
			<td>LB BROTH (LENNOX), Liquid Autoclave Sterilized</td>
			<td>BioShop Canada</td>
			<td>LBL666</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin, 900 &#181;g/mg</td>
			<td>Alfa Aesar by Thermo Fisher Scientific</td>
			<td>J66040</td>
			<td>It is recommended to perform a minimal inhibitory concentration (MIC) test for every batch made to ensure quality control of antimicrobial potency</td>
		</tr>
		<tr>
			<td>Quorum Volocity 6.3</td>
			<td>Quorum Technologies</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software

Les biofilms sont des agrégats de micro-organismes qui dépendent d’une matrice autoproduite de substance polymère extracellulaire pour la protection et l’intégrité structurelle. L’agent pathogène nosocomial, Pseudomonas aeruginosa, est connu pour adopter un mode de croissance de biofilm, provoquant une infection pulmonaire chronique chez les patients atteints de mucoviscidose (FK). Le programme informatique COMSTAT est un outil utile pour quantifier les changements induits par les antimicrobiens dans l’architecture du biofilm de P. aeruginosa en extrayant des données à partir d’images confocales tridimensionnelles. Cependant, le fonctionnement standardisé du logiciel est moins souvent abordé, ce qui est important pour un rapport optimal du comportement du biofilm et une comparaison intercentrique. Ainsi, l’objectif de ce protocole est de fournir un cadre simple et reproductible pour quantifier les structures de biofilms in vitro dans diverses conditions antimicrobiennes via COMSTAT. La technique est modélisée à l’aide d’un isolat de P. aeruginosa de la mucoviscidose, cultivé sous forme de réplicats de biofilm et exposé à la tobramycine et à l’anticorps monoclonal anti-Psl, Psl0096. L’approche étape par étape vise à réduire l’ambiguïté de l’utilisateur et à minimiser le risque de négliger des étapes cruciales de traitement d’image. Plus précisément, le protocole met l’accent sur l’élimination des variations subjectives associées au fonctionnement manuel de COMSTAT, y compris la segmentation d’images et la sélection de fonctions d’analyse quantitative appropriées. Bien que cette méthode oblige les utilisateurs à passer plus de temps à traiter des images confocales avant d’exécuter COMSTAT, elle permet de minimiser l’hétérogénicité erronée des biofilms dans les sorties automatisées.

Un isolat de P. aeruginosa cultivé chez un patient infecté par la mucoviscidose est utilisé pour démontrer les forces de cette approche dans la quantification précise des changements induits par les antimicrobiens dans l’architecture du biofilm in vitro . Le flux de travail global de ce modèle est représenté à la figure 1. La procédure de traitement d’image et d’analyse COMSTAT dans ImageJ est illustrée à la figure 2. La <stro...

Watch this Scientific Journal Video about Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software at JoVE.com

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Les modifications induites par les antimicrobiens de l’architecture du biofilm de Pseudomonas aeruginosa diffèrent entre les isolats cliniques cultivés chez des patients atteints de mucoviscidose et d’infection pulmonaire chronique. Après la microscopie confocale, le logiciel COMSTAT peut être utilisé pour quantifier les variations de l’architecture du biofilm (p. ex., surface, épaisseur, biomasse) pour des isolats individuels afin d’évaluer l’efficacité des agents anti-infectieux.

Quantification des effets des antimicrobiens sur l’architecture des biofilms in vitro à l’aide du logiciel COMSTAT

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

The significance of this protocol is that it offers a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions. The main advantage of this technique is that it helps eliminate subjective variations associated with the manual operation of COMSTAT, which ultimately facilitates the standardization and transparency of experiments across research centers. To image the Pseudomonas aeruginosa cultures, place the plate onto the stage of a confocal microscope, equipped with the appropriate laser excitation wavelengths and filter sets.And use a 20 to 25x water immersion lens to capture at least six layered z-stack images from the substratum to the top of each biofilm segment in 0.3 micrometer increments. Then save the images as OME-TIFFs for COMSTAT analysis. When all the images have been acquired, open the COMSTAT2 package in ImageJ and create a source folder on the desktop.Add a single OME-TIFF to the folder and open OME-TIFF from the source folder. Delete any empty layers containing no biomass. Select File, Import, and Image Sequence to import the file into ImageJ.Locate and highlight the source folder without opening it and click Select. A sequence options window will appear, click OK.Select Image, Transform, and Flip Z to flip the orientation of the biofilm so that the substratum is at the top of the stack. To define the image properties, select Image and Properties, a source window will appear.Specify the unit of length as micron, define the voxel depth as 0.3, and click OK.Select Image, Adjust, and Threshold. To adjust the image threshold in the threshold window, position the lower slider to the far right to manually set the maximum threshold value and use the upper slider to set the minimum threshold. Then use the slider in the image window to scroll through each layer to ensure that the background noise is sufficiently removed throughout the stack, and select Set to fix the lower threshold value.In the set threshold levels window, select OK, and set and fix the maximum threshold value similarly. Select Apply, click OK in the convert stack to binary window that appears, and exit the threshold window. Select Plugins, Bio-Formats, and Bio-Formats Exporter to save the file with a new file name in the source folder.A Bio-Formats Exporter multiple files window will appear, select OK.A Bio-Formats Exporter options window will appear, select OK again. To run COMSTAT, select plugins and COMSTAT2, an about window will appear, select OK.In the Observed Directories window, select Add, locate and highlight the source folder, highlight and click Choose. An Images in Directories window will appear that lists the OME-TIFFs to be analyzed.On the COMSTAT2.1 window, deselect Automatic Thresholding Otsu's Method and Connected Volume Filtering to ensure that the software uses preset threshold values for individual OME-TIFFs. In the COMSTAT2.1 window, select Biomass, Thickness Distribution and Surface Area, and click Go to run the program. The output data will be shown in the log window.The measurements will be automatically saved as TXT files in the source folder. In this representative analysis, a Pseudomonas aeruginosa isolate, cultured from an infected cystic fibrosis patient, was used to demonstrate the strengths of this approach, inaccurately quantifying antimicrobial induced changes in in vitro biofilm architecture. Live cell imaging can also visualize changes in Pseudomonas aeruginosa biofilm structure before and after biofilm specific monoclonal and tobramycin treatment.COMSTAT data analysis revealed significant differences between treated biofilm structures compared to control cultures. In tobramycin-treated cultures, a clear reduction in the average thickness and biomass was observed. However, in the presence of antibodies, the P.aeruginosa biofilm was resistant to these tobramycin-induced structural changes.When examining the surface to bio volume ratio, a significant reduction was observed in the presence of antibodies in both tobramycin-exposed and unexposed biofilms, indicating the formation of aggregates in antibody treated cultures. This procedure can be performed to quantitatively assess or compare the effects of different antimicrobials used to disrupt biofilm matrices. Using this technique as a general framework, researchers can explore different ways to refine the protocol to include new mathematical models and algorithmic plugins in ImageJ to better depict biofilm heterogenecy.

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Research

JoVE Journal

Immunology and Infection

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

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

Dans la formation de biofilms in vitro dans une diapositive Chambre de 8 puits

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

Recherche

Immunologie et infection

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.

Mesure des effets sur la fonction cellulaire anticorps de cardiomyocytes isolés

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

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.

Étude des effets des probiotiques sur la colonisation pneumococcique l&#39;aide d&#39;un<em&gt; In Vitro</em&gt; Respect de dosage

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.

Analyser les effets des cellules stromales sur le recrutement des leucocytes de débit

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

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Les analyses qualitatives et quantitatives pour la détection et la caractérisation des antimicrobiens en protéines

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In vitro</em&gt; Modèle de la barrière hémato-encéphalique utilisant la spectroscopie d&#39;impédance: un regard sur T Cell-endothélial cellulaire Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

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.

Cytométrie en flux Analyse de la particule liée Bet v 1 Allergène en 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 ...

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.

Visualisation des effets des expectorations sur le développement du biofilm Utilisation d&#39;un modèle Chambered lamelle couvre-objet

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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantifier la chimiotaxie Monocyte humain In Vitro et lymphocytes murins traite In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualisation de la Formation de biofilms Candida albicans à l’aide d’un dispositif automatisé de microfluidique

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...