Name: isolation, processing and analysis of murine gingival cells
Uploaded: 2013-07-02T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Questa ricerca è stata sostenuta da sovvenzioni dal Israel Science Foundation (n. 1418/11) per AHH e (n. 1933/12) a AW, la Fondazione israeliana tedesco per i giovani ricercatori (GIF Young) di AHH, e la dottoressa mi . Cabakoff Research Fondo di dotazione presso la University-Hadassah School of Dental Medicine di AHH e AW ebraico.

Preparare in anticipo: <ul><li> PBS + 2% FCS </li><li> PBS + 2% FCS con 2 mg / ml di collagenasi di tipo II e 1 mg / ml di DNAsi tipo I (1 ml per campione) </li><li> Strumenti chirurgici sterilizzati </li><li> 0,5 M soluzione di EDTA </li></ul> 1. Tomaia gengivale Tecnica escissione <ol><li> Euthanize topo, utilizzando una guida IACUC approvato. </li><li> Tagliare entrambi i lati del cavo orale tra le guance e il ramo mandibolare con un secco / smussato forbici dritte. </li><li> Tirare verso il basso la mandibola. </li><li> Tagliare i tessuti molli e duri in una linea immaginaria 1 millimetro dietro i molari terzi con forbici normali. Dirigono le forbici perpendicolari al piano dell&#39;osso palatale (Figura 2A, linee blu). </li><li> Incidere i tessuti molli e duri 2 millimetri dietro gli incisivi (Figura 2A, linee blu). </li><li> Tirare verso il basso la parte anteriore della bocca. La cavità nasale è osservabile dopo questo passaggio. </li><li> Posizionare le forbiciparallelo al palato, mettere una lama nella cavità nasale, l&#39;altra lama deve incidere al vestibolo. Tagliare finché l&#39;incisione posteriore (Figura 2A, linee verdi). Ripetere questo passaggio su entrambi i lati del mascellare superiore. Alla fine del mascellare è staccata dal resto del cranio (Figura 2B). </li><li> Rimuovere la mascella, tagliarlo nella sutura centro (Figura 2, nero linea tratteggiata) e tagliare il tessuto palatale di ciascun emi-mascellare fino a raggiungere l&#39;osso alveolare. </li><li> Sbucciare i tessuti gengivali (sia emi-mascellari) dal loro margine anteriore con Adson pinza (senza denti). </li><li> Posizionare la gengiva asportato in un piatto con PBS + 2% FCS. </li></ol> 2. Gengivale Elaborazione <ol><li> Mettere la gengiva asportato in un piatto di coltura tissutale (35 x 10 mm) con 1 ml di PBS + 2% FCS, 2 mg / ml di collagenasi di tipo II e 1 mg / ml di DNAsi Tipo I. </li><li> Tritare bene il tessuto con un 15 ° lama chirurgica sterile n. </li><li> Transfer il tessuto dalla piastra ad una provetta ml conica 15. </li><li> Lavare le cellule del tessuto / restano sulla piastra con 1 ml di PBS + 2% FCS e trasferimento alla stessa provetta. </li><li> Incubare in un agitatore incubatore per 20 minuti a 37 ° C, 200 rpm (raccomandazione: preriscaldare l&#39;incubatrice). </li><li> Aggiungere 20 ml di EDTA 0,5 M e incubare per altri 10 min a 37 ° C, 200 rpm. </li><li> Riempire la provetta fino a 12 ml con PBS + 2% FCS e centrifugare a 4 ° C, 400 x g per 8 minuti (si consiglia di pre-raffreddamento della centrifuga). </li><li> Rimuovere le cellule surnatante e risospendere con 2 ml di PBS + 2% FCS. </li><li> Filtrare il campione con un 70 micron cella-filtro e salvare il flusso attraverso. </li><li> Centrifugare a 4 ° C, 320 xg per 5 min. </li><li> Rimuovere le cellule surnatante e risospendere con 300 microlitri di PBS + 2% FCS. </li><li> Contare le cellule (circa 5-10 x 10 5 cellule sono attesi da un singolo mascellare). </li></ol> 3. Anticorpo extracellulare e intracellulareLa colorazione per Citometria a Flusso Analisi Si consiglia di lavorare all&#39;interno di una cappa, con le luci spente. È fondamentale per mantenere le cellule in ambiente freddo costante, così come tutti i materiali necessari. <ol><li> Colorare le cellule contro molecole extracellulari aggiungendo 0,2-0,5 ug di ciascun anticorpo selezionata per 1 x 10 6 cellule in un volume totale di 100 microlitri. </li><li> Vortex brevemente. </li><li> Incubare le provette per 15 minuti al buio a 4 ° C. </li><li> Lavare le cellule con 2 ml di PBS + 2% FCS. </li><li> Centrifugare a 4 ° C, 320 xg per 5 min. </li><li> Aspirare il surnatante e risospendere le cellule aggiungendo, goccia a goccia, 500 microlitri freddo BD Cytoperm / Cytofix mentre vortex. </li><li> Incubare le provette per 40 min a 4 ° C al buio. </li><li> Vortex brevemente. </li><li> Lavare le cellule due volte con 1 ml di freddo BD Perm / tampone di lavaggio e centrifuga a 800 xg per 7 minuti a 4 ° C. </li><li> Aspirare il surnatante eccetto 300 microlitri. </li><li> Colorare le cellule intracellulare dallaaggiungendo 1 ug di ciascun anticorpo selezionata per 1 x 10 6 cellule in un volume totale di 100 microlitri. </li><li> Vortex brevemente. </li><li> Incubare per 30 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 1 ml di freddo BD Perm / tampone di lavaggio e centrifuga a 800 xg per 7 minuti a 4 ° C. </li><li> Aspirare il surnatante e risospendere le cellule con l&#39;aggiunta di 300 ml freddo BD Perm / Wash Buffer. </li><li> Vortex brevemente. </li><li> Trasferire la sospensione, mentre il filtraggio di tubi FACS. </li><li> Tenere i tubi in un ambiente buio e fredda fino all&#39;analisi. </li></ol>

<ol>
	<li>Arizon, 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=Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss.">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li><li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, R., 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=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., 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=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

Gli autori non hanno nulla da rivelare.

Maxilla tessuti gengivali ottenuti da un singolo mouse sono sufficienti per analizzare sottopopolazioni di linfociti T e B, così come la loro capacità di esprimere molecole extracellulari ed intracellulari come noi descritta 1. Tuttavia, se le popolazioni di cellule rare sono di interesse (ad esempio DC), si raccomanda di tessuti piscina 2-3 topi. Di nota, se preferibile, è possibile sbucciare entrambi i tessuti palatali e gengivale e quindi di asportare la gengiva (una modifica del passo 1.8-1.9). Va rico...

Gengiva è il tessuto molle che circonda la porzione cervicale dei denti e copre il processo alveolare (Figura 1). La gengiva è un tipo di mucosa masticatoria che può essere ulteriormente separato in epitelio mucoso e tessuto connettivo (noto anche come sottomucosa o lamina propria). La struttura anatomica della gengiva e denti adiacenti permette ai batteri di risiedere e sviluppare placche (biofilm) che sfida costantemente il sistema immunitario locale. Come risultato, risposta infiammatoria sviluppa nella gengiva, che in talune circostanze diventa distruttiva - una condizione nota come malattie periodontali 2. In sostanza, patologie parodontali placca indotte possono essere suddivisi in gengivite e parodontite. La gengivite rappresenta una condizione reversibile della risposta infiammatoria locale che si limita alla gengiva. Periodontite, d&#39;altra parte, è un processo distruttivo irreversibile in cui l&#39;apparato di attacco (osso alveolare, parodontalelegamento, cemento e gengiva) è distrutto 3. La gengiva è stato proposto per servire siti sia come effettori e induttivi durante malattie parodontali 4. Studi sull&#39;uomo hanno suggerito che in risposta alla placca dentale, le cellule immunitarie effettrici e le molecole infiltrate o di partenza la gengiva 5-7 dinamicamente. Questa attività ha dimostrato di svolgere un ruolo importante nella distruzione parodontale 8,9. Considerando che i dati generati da questi studi hanno fornito informazioni importanti per quanto riguarda questo processo patologico, lavorando con i tessuti umani possiedono grandi limiti etici, tecnici e sperimentali. Lo sviluppo di modelli sperimentali animali sperimentazioni di causa-effetto con l&#39;impiego topi transgenici e negli interventi in vivo 10. Come risultato, le nostre conoscenze sui meccanismi coinvolti nella malattia parodontale è aumentato notevolmente nel corso degli ultimi due decenni. Anche così, a causa della complessità delle malattie periodontali, c&#39;èun dibattito in corso in merito alla natura della risposta immunitaria locale facilitando la distruzione dei tessuti. Vi è anche una mancanza nella nostra comprensione sulla funzione delle cellule immunitarie centrali nella gengiva durante malattie periodontali. E &#39;quindi essenziale studiare eventi infiammatori patologici che si verificano nel tessuto bersaglio della malattia, la gengiva.

<table><tbody><tr><td>Collagenase Type II</td><td>Worthington Biochemical Corp.</td><td>CLS-2</td><td></td></tr><tr><td>DNAse I</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>EDTA</td><td>SIGMA</td><td>E6758-100G</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>Biological Industries</td><td>04-121-1</td><td>Heat Inactivated</td></tr><tr><td>Vacuum-Driven Filter</td><td>Jet Biofil</td><td>FPE-204-500</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>SPL Lifesciences</td><td>93070</td><td></td></tr><tr><td>Tissue Culture Dish 35&amp;#215;10 mm</td><td>NUNC</td><td>153066</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>Anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD45.2 antibody</td><td>Biolegend</td><td>109819</td><td>Clone 104</td></tr><tr><td>Anti-mouse CD4 antibody</td><td>Biolegend</td><td>100413</td><td>Clone GK1.5</td></tr><tr><td>Anti-mouse CD8a antibody</td><td>Biolegend</td><td>100733</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>Biolegend</td><td>100214</td><td>Clone 17A2</td></tr><tr><td>Anti-mouse CD326 (Ep-CAM) antibody</td><td>Biolegend</td><td>118219</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse CD207 (Langerin) antibody</td><td>Imgenex</td><td>DDX0362D</td><td>Clone 929F3.01</td></tr><tr><td>Anti-mouse I-A&lt;sup&gt;d&lt;/sup&gt; (MHC-II) antobody</td><td>Biolegend</td><td>115010</td><td>Clone 39-10-8</td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo Software v 7.6.5</td><td>Tree Star</td><td></td><td></td></tr></tbody></table>

isolation, processing and analysis of murine gingival cells

Abbiamo sviluppato una tecnica per isolare e processare murino tessuto gengivale per la citometria a flusso e studi molecolari con precisione. La gengiva è un tessuto unico e importante per studiare i meccanismi immunitari perché è coinvolto nella risposta immunitaria contro i biofilm orale che potrebbe causare malattie parodontali. Inoltre, la vicinanza della gengiva al tessuto osseo alveolare consente anche studiando rimodellamento osseo in condizioni infiammatorie. Il nostro metodo consente di ottenere grandi quantità di cellule del sistema immunitario che permette l&#39;analisi anche di popolazioni di cellule rare, come le cellule di Langerhans e le cellule T regolatorie, come abbiamo dimostrato in precedenza 1. Impiegando i topi per studiare le risposte immunitarie locali coinvolti nella perdita di osso alveolare durante la malattia parodontale è vantaggioso a causa della disponibilità di vari strumenti immunologici e sperimentali. Tuttavia, a causa della loro piccola dimensione e l&#39;accesso relativamente scomodo per la gengiva murino, molti studi evitati esame di this tessuto critico. Il metodo descritto in questo lavoro potrebbe facilitare l&#39;analisi gengivale, che si spera di aumentare la nostra sottovalutare sul sistema immunitario orale e il suo ruolo durante le malattie parodontali.

Esempi di analisi di citometria a flusso su cellule gengivali sono presentati. Cellule gengivali pool da 2 topi sono stati eseguiti in un LSR II citofluorimetro e analizzati utilizzando il software FlowJo. Figura 3A dimostrato la distribuzione delle cellule gengivali da topi naive in un side scatter (SSC) rispetto forward scatter (FSC) trama. Strategia di gating per identificare (i) linfociti (ii) monociti / cellule dendritiche e (iii) granulociti è indicata. Fini confronto, presentiamo anche una trama...

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

Questo studio descrive una tecnica efficiente per isolare e processare tessuti gengivali dalla cavità orale topo per produrre una coltura cella singola. Le cellule risultanti possono essere ulteriormente utilizzati per l&#39;analisi di citometria a flusso e studi molecolari.

Isolamento, elaborazione ed analisi dei murini gengivali

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

isolation-processing-and-analysis-of-murine-gingival-cells

Research

JoVE Journal

Immunology and Infection

18.8K Views.  Hebrew University - Hadassah Medical Center. Questo studio descrive una tecnica efficiente per isolare e processare tessuti gengivali dalla cavità orale topo per produrre una coltura cella singola. Le cellule risultanti possono essere ulteriormente utilizzati per l'analisi di citometria a flusso e studi molecolari.

Video: Isolation, Processing and Analysis of Murine Gingival Cells

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Ricerca

Immunologia e infezioni

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Isolamento rapido di singole cellule da denti di topo e umani

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Biologia dello sviluppo

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento di cellule staminali del mouse ghiandole salivari

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biologia

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

Robust osufilazione modello di Murine Periodontitis per la valutazione dei neutrofili orali

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

Isolamento e coltura di cellule epiteliali gengivali umane primarie utilizzando Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Bioingegneria

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissezione e isolamento della glia murina da più regioni del sistema nervoso centrale

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Neuroscienze

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolamento delle cellule Miepiteliali da adulti murina lacrimale e ghiandole sottomandibolari

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

Un test di infezione batterica per lo studio delle interazioni batteri-cellule ospiti anaerobiche

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...