Name: isolation, characterization and functional examination of the gingival immune cell network
Uploaded: 2016-02-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

Todos los procedimientos experimentales descritos en este protocolo siguen las pautas requeridas y fueron aprobados por el Cuidado de Animales institucional y el empleo, NIDCR / NIH. 1. Prepárese por adelantado <ol><li> Preparar medio completo: RPMI suplementado con 2 mM L-glutamina, 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina y 10% de FBS. </li><li> Preparar medios DNasa: 50 ml de medio RPMI suplementado con 7,5 mg de DNasa (Haga fresco, mantener en hielo en todo momento). </li><li> Prepar...

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La técnica actual produce con éxito un gran número de células inmunes (de un solo ratón) adecuado, no sólo para la caracterización fenotípica, sino también para estudios funcionales ex vivo. Otro grupo había publicado previamente un protocolo para aislar y caracterizar células inmunes gingivales murino y se introduce el valor de la utilización de la citometría de flujo multicolor en el estudio de la periodontitis en modelos animales 9. Tras la resolución de problemas considerables la mo...

Tejidos gingivales rodean la dentición humana y murina y están constantemente expuestos al complejo biofilm del diente 1. La red de células inmunes la vigilancia de la barrera gingival es vital para mantener la integridad del tejido, asegurando homeostasis con los microbios comensales locales y, al mismo tiempo, proporcionando una inmunidad eficaz contra el desafío patogénico 2. Para lograr la homeostasis, el sistema inmunológico está cuidadosamente adaptada al entorno gingival creación de una red de células inmunes altamente especializado, sin embargo, poco se conoce el detalle de las poblaciones de células inmunes gingivales y su papel en el mantenimiento de la inmunidad tisular 2. Cuando se interrumpe la homeostasis inmune en la encía, ya sea mediante el aumento de la susceptibilidad y / o presencia de comunidades microbianas dysbiotic, una condición inflamatoria del huésped, surge la periodontitis 3 4. La periodontitis es una enfermedad inflamatoria común, lo que lleva a la pérdida del diente sPOYO estructuras. En sus formas severas se observa en aproximadamente el 10% de la población general 5. La disección de los factores clave implicados en la susceptibilidad periodontitis y la progresión ha sido difícil 6. Sin embargo, los modelos animales han sido extremadamente útiles en la comprensión de los mecanismos de iniciación y progresión periodontitis 7. Los modelos pueden ser empleados para definir las poblaciones de células y mediadores moleculares clave que son vitales para el mantenimiento de la homeostasis inmune y el desarrollo de la unidad de la periodontitis. Tal visión va a transformar nuestra comprensión del control-encía específica de la homeostasis inmune y mejorar nuestra comprensión actual de la patogénesis de la enfermedad.

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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.

Para ilustrar la aplicación del protocolo, mostramos los resultados representativos que examinan la red de células inmunes en la encía de los ratones con y sin periodontitis (WT vs. LFA - / -, Figura 2A - C). FACS representativos gráficos muestran células CD45 + hematopoyéticos vivos en la encía (Figura 2A, 2C). El aislamiento y la transformación de las células inmunes con este protocolo da...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

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

Hemos establecido una técnica para el aislamiento, la caracterización fenotípica y análisis funcional de células inmunes de encía murino.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

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

The overall goal of this method is to isolate gingival immune cells for the in-depth characterization of the immune responses related to oral immunity and periodontitis. This method can help answer key questions in the field of oral immunity, such as what cellular factors are implicated in the pathogenesis of periodontitis. The main advantages of this technique are that it allows the evaluation of the oral immune cell network and the isolation of enough viable cells for downstream in-vitro investigations.As a first step of this protocol, begin by placing pins to immobilize the body of the mouse and open the thoracic and abdominal cavities to expose the heart and internal organs. Next, make an incision in the vena cava, and use a 3-milliliter syringe, equipped with a 27-gauge needle, to profuse 3 milliliters of PBS via the left ventricle. When all of the blood has been flushed, pin the head of the mouse onto a dissecting pad, with the stomach facing up.Then use scissors to cut each commissure of the lip towards the neck, separating the skin that covers the mandible and neck region, and exposing the underlying tissues and muscles. Cut between the lower incisors to separate both sides of the mandible. Immobilize each side of the separated mandible with 25-gauge needles to access the oral cavity.After identifying the palate, use a number 10 surgical blade to make vertical incisions just anterior to the first molar, and posterior to the third molar. Then dissect the tissue away from the nasal cavity, and also dissect the blocks from the mandible. Then eliminate the excess tissue from the border of the gingiva and transfer the maxillary and mandibular blocks into a 50-milliliter conical tube containing 5 milliliters of collagenase and DNase on ice.Digest the tissue in a shaker incubator at 37 degrees Celsius for one hour, adding 50 microliters of 0.5 molar EDTA during the last five minutes. Then add 5 milliliters of cold DNase medium to the tissues, and gently mix the tube contents by swirling. Next, transfer the four blocks of tissue into a petri dish.And cover them with 500 microliters of fresh DNase medium. Use a scalpel to remove the gingiva from each block of tissue. And transfer the entire contents of the dish into a 70 micron cell strainer on top of a 50-milliliter tube.Wash the petri dish and strainer with 3 to 5 milliliters of fresh DNase medium. And then use the plunger of a sterile, 3-milliliter syringe to mash the gingival tissue against the mesh of the strainer. Rinse the strainer with 30 to 35 millilters of cold DNase medium, and centrifuge the filtrate.Then resuspend the palate in 1 milliliter of complete medium, and determine the number of viable cells. The isolation and processing of immune cells as just demonstrated, yields a sufficient number of cells to perfom an ex-vivo stimulation and characterization of the gingival cell cytokine secretion patterns from both wild-type, as well as periodontitis-susceptible animals. Indeed, the murine gingival immune cell composition can be analyzed as demonstrated in these density plots evaluating the t and natural killer cell compartments in periodontitis-susceptible LFA knockout mice.Using the appropriate gating strategies, the intracellular cytokine production of TCR beta CD4 positive, TCR beta CD8 positive, and TCR gamma delta positive t cells, as well as NK, and innate lymphoid cells, can also be determined. Once mastered, the cells can be isolated in three hours for immunostaining if the technique is performed properly. While attempting this procedure, it's important to remember to complete all of the steps efficiently and quickly, keeping the cells on ice whenever they are not being manipulated.Following this procedure, other methods, like cell sorting, can be performed to answer additional questions related to the function of the specific cell subsets.

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Aislamiento de tejido adiposo células inmunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Un<em&gt; In Vitro</em&gt; Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Aislamiento y caracterización de micropartículas de neutrófilos derivado de estudios funcionales

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Aislamiento de mastocitos de peritoneo derivado y su caracterización funcional con Ca2 +-proyección de imagen y pruebas de degranulación

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...