Introdução ao Equipamento <ol><li> Tecido é cuidadosamente dissociada, utilizando tubos de violeta C em uma única célula viável suspensões. </li><li> O PBS / BSA-tampão de EDTA é utilizado para dissociar as células. O buffer é preparada por adição de 1 parte de solução de BSA estoque para 20 partes de autoMACS lavagem solução contendo EDTA. </li><li> O MACS pré-separação Filtros são usados ​​para que as amostras de tecido dissociado estão livres de todos os aglomerados de células, promovendo a separação MACS sucesso e posterior análise. </li></ol> Dissociar as células do baço <ol><li> Baços todo são disscociated em uma única célula suspensões de esplenócitos usando o Dissociator gentleMACS. </li><li> Para iniciar o procedimento, coloque o tubo C com um volume de tampão de acordo com a massa do baço. Um baço saudável de um jovem adulto camundongo BALB / c fêmeas pesa cerca de 80-120 mg. Cada 1 a 2 baços dessa massa necessitam de 3 mL de tampão. Baços de animais mais velhos ou animais com infecções são muitas vezes maiores e devem ser pesados ​​em buffer. Os volumes de buffer deve ser adaptado em conformidade. Não exceder o volume total do buffer de 10 mL. </li><li> Adicione o baço dissecados para o tubo C contendo 3 ml de tampão. </li><li> Certifique-se de que a tampa do tubo C é apertado e conecte o tubo C ao dissociator com a tampa para baixo. </li><li> Ligue o dissociator. Por 1-2 baços, selecione m_spleen_01 programa. O programa terá 56 segundo para ser concluído. Se o número de baços é maior, usar o programa m_spleen_04; este programa pode ser usado para até 720 mg de tecido esplênico. </li><li> Após a conclusão do programa, o tecido do baço nativa é dissociada em uma suspensão esplenócitos. </li><li> Girar a suspensão de células inteiras em 300xg em temperatura ambiente por cerca de 30 seg. Isso irá garantir que o rotor eo estator são libertados de todas as células que são coletados na parte inferior do tubo C. </li><li> Retirar a amostra através do septo, selada abertura da tampa do tubo C usando uma pipeta 1000 microlitro. </li></ol> Etapa de filtração <ol><li> Para começar a etapa de filtração, aplicar o tecido dissociado para o Filtro de pré-separação ou um filtro de células com 30 mM tamanho dos poros. O filtro deve caber um tubo de 15 mL por 1-2 baços ou um tubo de 50 mL por mais de 2 baços. </li><li> Deixe a suspensão de células passar. Em seguida, lave o filtro através da aplicação de 5 mL de tampão. </li><li> Agora, centrifugar o tubo com a suspensão de células em 300xg, em temperatura ambiente por 10 minutos para coletar todos os esplenócitos em um baço pellet celular. </li><li> Agora, que temos uma pelota de esplenócitos dissociados, devemos aspirar o sobrenadante e ressuspender o pellet celular em uma quantidade adequada de buffer. </li></ol> Dissociação baço usando o Dissociator gentleMACS renderá uma elevada percentagem de esplenócitos viável, o que é demonstrado por citometria de fluxo usando o Analisador MACSQuant. Células mortas estão manchadas pelo iodeto de propídio. No exemplo fornecido, o atacante lado gráfico de pontos de dispersão mostra o esplenócitos preparado.

Todos os protocolos e fichas de dados estão disponíveis em<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

Para evitar dissociações manuais entediantes e muitas vezes doloroso, o Dissociator gentleMACS permite dissociar o tecido de forma muito eficiente sob condições controladas e reproduzíveis. O tecido procedimento de dissociação é suave para as células e suspensões colheitas de células com uma taxa alta viabilidade. O Dissociator gentleMACS em combinação com C Tubes é projetado para a preparação de uma única célula suspensões de vários tecidos. O instrumento é equipado com uma série de programas pré-definidos para dissociar baço mouse, fígado, pulmão, ou tecido neural. A gama de programas e protocolos será continuamente ampliado para incluir outros tecidos.

<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

De uma única célula suspensões são um pré-requisito para experimentos na separação de células, a análise de células e cultura de células. Para evitar dissociações manual tedioso e doloroso o Dissociator gentleMACS permite dissociar o tecido de forma muito eficiente sob condições controladas e reproduzíveis. O Dissociator gentleMACS pode perfeitamente dissociar baço mouse, combinando economia de tempo e padronização com segurança do usuário. Este vídeo descreve como dissociar baços mouse usando o Dissociator gentleMACS, um dispositivo de bancada automatizado que pode atrapalhar mecanicamente tecidos utilizando tubos especiais para produzir suspensões de células viáveis. Após a dissociação, baços são filtradas, centrifugadas e ressuspendidas para outras aplicações.

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 vídeo descreve uma técnica simples e que economiza tempo para a dissociação de tecidos automatizado utilizando o Dissociator gentleMACS para preparar uma única célula suspensões de esplenócitos mouse.

Preparação de uma única célula Suspensões de Spleen rato com o Dissociator gentleMACS

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

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

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JoVE Journal

Biology

37.7K visualizações.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

Vídeo: 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.

Preparando-se Fator de Crescimento de Células T esplenócitos Rat

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. ...

Biologia

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.

Preparação padronizado de uma única célula Suspensões do tecido pulmonar do rato usando o Dissociator 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.

Geração de Single-Cell Tissue Suspensões de rato 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

A citometria de fluxo para avaliar o estado de cromatina em 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 ...

Imunologia e Infecção

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.

Isolamento de células spermatogênicas murinas usando um corante de ligação de DNA permeável celular-permeável violeta

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

Biologia do Desenvolvimento

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.

Agregação celular encapsulada da matriz da membrana basal para investigação da formação do tecido do baço 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.

Isolamento e transferência adotiva de sal alta trataram apresentadoras de células dendríticas

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.

Caracterização citométrica de fluxo do desenvolvimento 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 refinamento para isolamento de exossomos derivados do baço 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>Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

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

Isolamento e caracterização de exossomos derivados de tecidos de baço de camundongo

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

Preparação de uma única célula Suspensões de Spleen rato com o Dissociator gentleMACS

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