Introducción a los Equipos <ol><li> Tejido es ligeramente disociado, con violeta tubos C en viable una sola célula suspensiones. </li><li> El tampón PBS / BSA EDTA se utiliza para disociar las células. El buffer se prepara añadiendo una parte de la solución de archivo BSA a 20 partes de autoMACS Solución de enjuague que contiene EDTA. </li><li> El MACS separación pre-filtros se utilizan para que las muestras de tejido disociado están libres de todos los grupos de células, la promoción de la separación MACS éxito y su posterior análisis. </li></ol> Disociar las células del bazo <ol><li> El bazo entero se disscociated en una sola célula suspensiones de esplenocitos con el disociador gentleMACS. </li><li> Para comenzar el procedimiento, el tubo de carga C con un volumen de tampón de acuerdo a la masa del bazo. Un bazo sano de un adulto joven hembra BALB / c ratón pesa alrededor de 80-120 mg. Cada 1 a 2 bazos de esta masa requiere de 3 ml de solución tampón. El bazo de los animales más viejos o los animales con infecciones suelen ser mayores y se deben sopesar en el buffer. Los volúmenes de almacenamiento intermedio debe ser reducido en consecuencia. No excedan de un volumen total de memoria intermedia de 10 ml. </li><li> Añadir el bazo diseccionados para el metro C con 3 ml de solución tampón. </li><li> Asegúrese de que la tapa del tubo C es apretado y conectar el tubo de C a la disociador con la tapa hacia abajo. </li><li> Encienda el disociador. Por 1-2 el bazo, seleccione m_spleen_01 programa. El programa tendrá 56 segundos para completar. Si el número de bazo es mayor, use el programa m_spleen_04, este programa puede ser utilizado para un máximo de 720 mg de tejido esplénico. </li><li> Después de completar el programa de los tejidos del bazo nativos se disocia en una suspensión de esplenocitos. </li><li> Centrifugar la suspensión de células enteras en 300xg a temperatura ambiente durante unos 30 segundos. Esto asegurará que el rotor y el estator se libera de todas las células que se recogen en la parte inferior del tubo C. </li><li> Retire la muestra a través del tabique sellado de apertura en la tapa del tubo con una pipeta de C 1000 microlitros. </li></ol> Filtración de paso <ol><li> Para comenzar la etapa de filtración, aplicar el tejido disociado al filtro de pre-separación o un colador de células con un tamaño de micras de poro 30. El filtro debe ajustarse a un tubo de 15 ml por 1-2 el bazo o un tubo de 50 ml por más de 2 bazos. </li><li> Deje que la suspensión de células pasan a través. A continuación, lavar el filtro mediante la aplicación de 5 ml de solución tampón. </li><li> Ahora, centrifugar el tubo con la suspensión celular a 300xg, a temperatura ambiente durante 10 minutos para recoger todos los esplenocitos en un pellet de células del bazo. </li><li> Ahora, que tenemos una bolita de esplenocitos disociado, que debe aspirar el sobrenadante y resuspender el botón celular en una cantidad adecuada de tampón. </li></ol> La disociación del bazo con el disociador gentleMACS dará un alto porcentaje de esplenocitos viables, lo cual es demostrado por citometría de flujo utilizando el analizador de MACSQuant. Las células muertas se tiñen con yoduro de propidio. En el ejemplo proporcionado, la dispersión frontal lado gráfico de puntos muestra los esplenocitos preparado.

Todos los protocolos y hojas de datos están disponibles en<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

Para evitar disociaciones manual tedioso ya menudo dolorosa, el disociador gentleMACS permite disociar los tejidos de manera muy eficiente en condiciones controladas y reproducibles. El procedimiento de disociación del tejido es suave con las células y las suspensiones de producción de células con una tasa alta viabilidad. El disociador gentleMACS en combinación con C Tubos está diseñado para la preparación de una sola célula suspensiones de varios tejidos. El instrumento está equipado con una serie de programas pre-establecidos para disociar de bazo de ratón, hígado, pulmón, o en el tejido neural. La gama de programas y protocolos serán continuamente expandido para incluir otros tejidos.

<table><tbody><tr><td>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx&lt;/a&gt;</td></tr><tr><td>13 mL Tubes</td><td>Miltenyi Biotec</td><td>130-091-596</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx&lt;/a&gt;</td></tr><tr><td>autoMACS&amp;#8482; Rinsing Solution </td><td>Miltenyi Biotec</td><td>130-091-222</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx&lt;/a&gt;</td></tr><tr><td>MACS&amp;#174; BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx&lt;/a&gt;</td></tr><tr><td>Pre-Separation Filters </td><td>Miltenyi Biotec</td><td>130-041-407</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&lt;/a&gt;</td></tr><tr><td>MACSQuant Analyzer</td><td>Miltenyi Biotec</td><td>130-092-197</td><td></td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>130-093-233</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&lt;/a&gt;</td></tr></tbody></table>

preparation of single-cell suspensions from mouse spleen with the gentlemacs dissociator

Una sola célula suspensiones son un requisito previo para realizar experimentos en la separación celular, el análisis de células y cultivos celulares. Para evitar disociaciones manual tedioso ya menudo dolorosa la disociador gentleMACS permite disociar los tejidos de manera muy eficiente en condiciones controladas y reproducibles. El disociador gentleMACS óptima se puede disociar de bazo de ratón, la combinación de ahorro de tiempo y de normalización por el usuario de seguridad. Este vídeo se describe cómo disociar el bazo del ratón con el disociador gentleMACS, un sistema automatizado de sobremesa dispositivo mecánico que puede alterar los tejidos que utilizan tubos especiales para producir suspensiones de células viables. Después de la disociación, el bazo se filtra, se centrifuga y se resuspende para otras aplicaciones.

Watch this Scientific Journal Video about Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator at JoVE.com

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Este video describe un simple ahorro de tiempo para la técnica de la disociación del tejido automatizado utilizando el disociador gentleMACS para preparar una sola célula suspensiones de esplenocitos de ratón.

Preparación de una sola célula suspensiones de bazo de ratón con el disociador gentleMACS

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

<a class="grey_button" href="/video/1045">
&#x4E2D;&#x6587;
</a>


<a class="grey_button" href="/video/1102">
JapaneseVersion
</a>

preparation-single-cell-suspensions-from-mouse-spleen-with-gentlemacs

Research

JoVE Journal

Biology

37.6K vistas.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

Video: Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

La preparación de Factor de Crecimiento Celular T a partir de esplenocitos de rata

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Investigación

Biología

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

La preparación estandarizada de un solo suspensiones de células de tejido pulmonar de ratón con el disociador gentleMACS

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Generación de un solo suspensiones de células de tejido de ratón Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

El uso de la citometría de flujo para evaluar el estado de la cromatina en las células T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Inmunología e Infección

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Aislamiento de células espermatogénicas murinas usando un tinte de unión de ADN permeable a células violetas-excitado

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Biología del Desarrollo

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

Agregación celular encapsulada en matriz de membrana basal para investigar la formación de tejido del bazo murino

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Aislamiento y la transferencia adoptiva de sal alta tratan células dendríticas presentadoras de antígenos

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Caracterización citométrica de flujo del desarrollo de células B murinas

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

<meta charset="utf8"></head><body>Técnicas de refinamiento para el aislamiento de exosomas derivados del bazo murino

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Preparación de una sola célula suspensiones de bazo de ratón con el disociador gentleMACS

Transcribir

Resumen

Explorar más videos

Capítulos en este video

La preparación de Factor de Crecimiento Celular T a partir de esplenocitos de rata

La preparación estandarizada de un solo suspensiones de células de tejido pulmonar de ratón con el disociador gentleMACS

Generación de un solo suspensiones de células de tejido de ratón Neural

El uso de la citometría de flujo para evaluar el estado de la cromatina en las células T

Aislamiento de células espermatogénicas murinas usando un tinte de unión de ADN permeable a células violetas-excitado

Agregación celular encapsulada en matriz de membrana basal para investigar la formación de tejido del bazo murino

Aislamiento y la transferencia adoptiva de sal alta tratan células dendríticas presentadoras de antígenos

Caracterización citométrica de flujo del desarrollo de células B murinas

Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón