Il lavoro è stato finanziato dalla dell&#39;NIDCD / NIH R01DC003915 a LO Bakaletz.

1. Nella formazione di biofilm in vitro <ol><li> Colonie batteriche (che sono state coltivate su agar appropriato durante la notte), sono stati sospesi nei media e la OD 490 è stato rettificato a 0,65 </li><li> La sospensione batterica risultante è stato poi diluito 1:6 (1 ml di sospensione batterica + 5 ml preriscaldata media) e incubate a 37 ° C con 5% di CO 2 per circa 3 ore per raggiungere la metà del log fase. </li><li> Diluire il mid-log 1:2500 sospensione di crescita con pre-riscaldato media e 200 ul luogo di diluizione in ciascun pozzetto di una 8-scivolo e da camera. </li><li> Incubare a 37 ° C con 5% di CO 2. </li><li> Dopo circa 16 ore, media aspirato da un angolo di ogni camera e aggiungere 200 microlitri fresco, pre-riscaldato a medio - lungo dispensare muro di camera in modo da non creare forze di taglio all&#39;interno della camera che potrebbe disturbare il biofilm. </li><li> Se incubazione più lungo di 24 ore, il cambiamento medio ogni 12 ore o secondo necessità per mantenere la vitalità dei batteri. </li></ol> 2. Visualizzazione dei biofilm <ol><li> Aspirare il terreno da ogni camera, come sopra descritto, e lavare biofilm residente due volte delicatamente con soluzione salina sterile. </li><li> Aggiungere 200 ml di BacLight Live / Dead macchia (3 microlitri componente A 3 componente B + microlitri per mL di soluzione salina) per ciascun pozzetto e incubare a temperatura ambiente per 15 minuti. Proteggere la cultura dalla luce da questo punto in avanti. </li><li> Aspirare la macchia e lavare due volte delicatamente con soluzione salina sterile come prima. </li><li> Aggiungere 200 ml di formalina neutra tamponata ad ogni pozzetto e incubare per 30 minuti a temperatura ambiente per risolvere campione. </li><li> Rimuovere fissativo e smaltire in conformità alle direttive istituzionali. </li><li> Lavare due volte con soluzione salina e smaltimento dei liquidi di lavaggio contenenti formalina come sopra. </li><li> Rimuovere i pozzi di plastica dalla diapositiva, aggiungere abbastanza salina in ogni pozzetto in modo che quando un coprioggetto è posto sulla guarnizione, non siano presenti bolle d&#39;aria. </li><li> Coprioggetto e sigillare i bordi del coprioggetto con mezzo di montaggio. Smalto per le unghie può essere utilizzato per sigillare le diapositive tuttavia le diapositive non sarà permanente. </li><li> Lasciare che il sigillante ad asciugare per circa 1 ora prima di analizzare tramite microscopia. </li></ol> 3. Risultati <img src="/files/ftp_upload/2481/2481fig1.jpg" alt="Figura 1" /> Figura 1. Rappresentante immagini 3D composito di un biofilm formati in una 8-scivolo e da camera. I batteri sono stati etichettati con Live / Dead macchia batteri in cui vivono i batteri fluorescenti verdi e morto fluorescenza rossa. (A) immagine composita di biofilm intera che mostra l&#39;architettura diversi con torri e canali d&#39;acqua. (B) biofilm Stessa (A), ora visto in sezione trasversale.

<ol>
	<li>Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+otitis+media:+evidence+and+relevance.">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, S17-S19 (2007).</li><li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li></ol>

Nessun conflitto di interessi dichiarati.

Comprendere il meccanismo con cui biofilm forma, così come caratterizzante la loro componenti della matrice, è un&#39;area di grande interesse. Questo protocollo può essere usato per aiutare a identificare le proteine ​​o altri costituenti che contribuiscono alla integrità strutturale del biofilm, così come aiutare a identificare e target le proteine ​​che possono si prestano ad una applicazione terapeutica. I tempi di incubazione e diluizioni nel protocollo sopra descritti sono stati stabiliti per la formazione di biofilm ottimale nontypeable da Haemophilus influenzae (NTHI). Alcuni lavori preliminari può essere necessaria per ottimizzare questi passaggi per altre specie di batteri (ad esempio diluizione iniziale e tempo di incubazione necessario per raggiungere la metà del log fase di crescita). La diluizione 1:2500 per la semina iniziale delle camere è stato creato per garantire che un biofilm robusta si svilupperebbe in camera di diapositive come indotta da questo organismo e nei tempi di incubazione ha dichiarato, senza il biofilm overgrowing la camera e / o estenuanti sostanze nutritive disponibili . Di conseguenza, la diluizione iniziale e tempi di incubazione possono o devono essere adeguate per le altre specie batteriche.

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<td>Chocolate II agar</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>B21267X</td>
<td>&nbsp;</td>
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<td>8-well chamber slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-656-18</td>
<td>&nbsp;</td>
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<td>BacLight Live/Dead bacterial viability kit</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9439023</td>
<td>&nbsp;</td>
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<td>BHI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>211059</td>
<td>&nbsp;</td>
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<td>Hemin</td>
<td>&nbsp;</td>
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<td>H5533</td>
<td>&nbsp;</td>
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<td>&beta;-NAD</td>
<td>&nbsp;</td>
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<td>N7004</td>
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in vitro biofilm formation in an 8-well chamber slide

La natura di molte malattie croniche è attribuito alla formazione di biofilm batterici che sono recalcitranti alla tradizionale terapia antibiotica. Biofilm sono comunità di batteri associati collegato a una superficie e racchiusi in una matrice. Il ruolo della matrice extracellulare è multiforme, anche facilitando l&#39;acquisizione di nutrienti, e offre una protezione significativa contro gli stress ambientali (ad esempio risposta immunitaria). Nel tentativo di acquisire una migliore comprensione di come i batteri in un biofilm rispondere agli stress ambientali che abbiamo usato un protocollo in cui noi visualizziamo biofilm batterici che si sono formati in un 8-scivolo e da camera. I biofilm sono stati colorati con la BacLight Live / Dead macchia ed esaminate con un microscopio confocale a caratterizzare la dimensione relativa biofilm, e la struttura in diverse condizioni di incubazione. Z-stack immagini sono state raccolte tramite microscopia confocale e analizzati da COMSTAT. Questo protocollo può essere utilizzato per contribuire a spiegare il meccanismo e cinetica con cui formare biofilm, oltre a identificare i componenti che sono importanti per la struttura biofilm e la stabilità.

Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

In vitro Biofilm Formation in an 8-well Chamber Slide

Questo articolo descrive la procedura per la formazione e la visualizzazione di un biofilm batterico cresciuto all&#39;interno di un 8-scivolo e da camera

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

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

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Research

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Immunology and Infection

In vitro Biofilm Formation in an 8-well Chamber Slide

40.0K Views.  The Research Institute at Nationwide Children's Hospital. Questo articolo descrive la procedura per la formazione e la visualizzazione di un biofilm batterico cresciuto all'interno di un 8-scivolo e da camera

Video: In vitro Biofilm Formation in an 8-well Chamber Slide

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.

Microtitolazione Dish Biofilm saggio Formazione

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

Ricerca

Immunologia e infezioni

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Enterico invasione batterica della cellule epiteliali intestinali<em&gt; In Vitro</em&gt; È drammaticamente migliorata utilizzando una camera di diffusione verticale Modello

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Misura High Throughput di extracellulare DNA di uscita e quantitativa NET Formazione in neutrofili umani<em&gt; In Vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Metodi in vitro per il confronto obiettivo vincolante e CDC induzione Tra terapeutiche Anticorpi: Applicazioni nel Analisi Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

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.

Visualizzazione della formazione di Biofilm in Candida albicans utilizzando un dispositivo microfluidico automatizzato

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

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formazione di Biofilm indotta da sali biliari in patogeni enterici: tecniche di identificazione e quantificazione

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Canini trappola extracellulari del neutrofilo formazione: Analisi dinamica e quantitativa mediante microscopia a fluorescenza

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...