Introduzione alla Attrezzature <ol><li> Il tessuto è leggermente dissociato, utilizzando viola Tubi C in singola cellula vitale sospensioni. </li><li> La PBS / BSA tampone EDTA è usato per separare le cellule. Il buffer è preparato con l&#39;aggiunta di 1 parte di soluzione madre BSA per 20 parti di autoMACS Risciacquo Soluzione contenente EDTA. </li><li> La pre-separazione filtri MACS sono usati in modo che i campioni di tessuto dissociate sono liberi di tutti i grumi di cellule, favorendo il successo separazione MACS e successiva analisi. </li></ol> Dissociare le cellule della milza <ol><li> Milze intere disscociated in cella singola sospensioni di splenociti utilizzando il dissociatore gentleMACS. </li><li> Per iniziare la procedura, caricare la metropolitana C, con un volume di tampone a seconda della massa della milza. Una milza sana di un giovane adulto femmina BALB / c del mouse pesa circa 80-120 mg. Ogni 1 o 2 milza di questa massa richiedono 3 ml di tampone. Milza di animali più vecchi o animali con infezioni sono spesso più grandi e devono essere valutati nel buffer. I volumi buffer deve essere ripartita in proporzione. Non superano un volume totale di buffer di 10 ml. </li><li> Aggiungere la milza sezionato per la metropolitana C contenente 3 ml di buffer. </li><li> Assicurarsi che il tappo del tubo C è stretto e collegare il tubo C al dissociatore con il tappo verso il basso. </li><li> Accendere il dissociatore. Per 1-2 milza, selezionare m_spleen_01 programma. Il programma avrà 56 secondi per essere completata. Se il numero di milza è maggiore, usare il programma m_spleen_04, questo programma può essere utilizzato per un massimo di 720 mg di tessuto milza. </li><li> Dopo il completamento del programma del tessuto milza nativa è dissociato in una sospensione splenocyte. </li><li> Spin down la sospensione a cellule intere, a 300xg a temperatura ambiente per circa 30 sec. Questo farà sì che il rotore e lo statore sono liberati di tutte le cellule che vengono raccolte sul fondo del tubo C. </li><li> Rimuovere il campione attraverso il setto-sigillato l&#39;apertura del tappo del tubo C con una 1000 pipetter microlitro. </li></ol> Filtrazione passo <ol><li> Per iniziare la fase di filtrazione, applicare il tessuto dissociato al pre-filtro di separazione o di un colino cellula con 30 micron dimensioni dei pori. Il filtro dovrebbe montare un tubo di 15 ml per 1-2 milza o un tubo da 50 ml per più di 2 milze. </li><li> Lasciate che la sospensione di cellule passano attraverso. Poi lavare il filtro applicando 5 ml di tampone. </li><li> Ora, centrifugare la provetta con la sospensione cellulare a 300xg, a temperatura ambiente per 10 minuti per raccogliere tutte splenociti in una milza pellet. </li><li> Ora, che abbiamo un pellet di splenociti dissociato, dobbiamo aspirare il supernatante e risospendere il pellet cellulare in una quantità adeguata di buffer. </li></ol> Dissociazione milza utilizzando il dissociatore gentleMACS produrrà una elevata percentuale di splenociti vitale, che è dimostrato mediante citometria di flusso utilizzando l&#39;analizzatore MACSQuant. Le cellule morte sono colorate con ioduro di propidio. Nell&#39;esempio fornito, l&#39;attaccante scatter plot mostra il lato dot splenociti preparato.

Tutti i protocolli e le schede tecniche sono disponibili all&#39;indirizzo<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

Per evitare di dissociazioni manuali ripetitive e spesso dolorosa, il dissociatore gentleMACS permette di dissociare i tessuti in maniera molto efficiente, in condizioni controllate e riproducibili. La procedura di dissociazione tessuto è delicato per le cellule e sospensioni cellulari produce un alto tasso di vitalità. Il dissociatore gentleMACS in combinazione con C Tubi è stata progettata per la preparazione di singola cellula sospensioni da vari tessuti. Lo strumento è dotato di una serie di programmi preimpostati per dissociare il mouse milza, fegato, polmone, o tessuto neurale. La gamma di programmi e protocolli sarà continuamente ampliata per includere altri tessuti.

<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

Monocellulari sospensioni sono un prerequisito per esperimenti di separazione delle cellule, l&#39;analisi delle cellule e coltura delle cellule. Per evitare di dissociazioni manuali ripetitive e spesso doloroso il dissociatore gentleMACS permette di dissociare i tessuti in maniera molto efficiente, in condizioni controllate e riproducibili. Il dissociatore gentleMACS può dissociarsi in modo ottimale milza del mouse, coniugando risparmio di tempo e di normalizzazione dall&#39;utente sicurezza. Questo video spiega come dissociare milza del mouse utilizzando il dissociatore gentleMACS, un sistema automatico da banco dispositivo in grado di distruggere meccanicamente i tessuti con tubi speciali per la produzione di sospensioni di cellule vitali. A seguito di dissociazione, milza vengono filtrati, centrifugati e risospesi per ulteriori applicazioni.

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

Questo video descrive un semplice, di risparmiare tempo tecnica per la dissociazione dei tessuti automatizzato mediante il dissociatore gentleMACS per preparare cella singola sospensioni di splenociti mouse.

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore 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

37.7K Views.  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.

Preparazione T fattore di crescita cellulare da splenociti 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. ...

Ricerca

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.

La preparazione standardizzata di Single-Cell Sospensioni da tessuto polmonare del mouse utilizzando il dissociatore 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.

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

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

L&#39;uso di citometria a flusso per valutare lo stato della cromatina in cellule T

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

Immunologia e infezioni

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 delle cellule spermatogeniche murine utilizzando un colorante legante del DNA permeabile alle cellule violetto

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

Biologia dello sviluppo

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.

Aggregazione cellulare incapsulata della matrice della membrana basale per lo studio della formazione del tessuto milza 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 trasferimento adottivo di sale alta trattati cellule dendritiche presentanti l'antigene

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.

Caratterizzazione citometrica a flusso dello sviluppo delle cellule B murine

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>Tecniche di affinamento per l'isolamento di esosomi derivati dalla milza murina

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 caratterizzazione di esosomi derivati da tessuti di milza di topo

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

Isolamento e caratterizzazione di esosomi derivati da tessuti di milza di topo

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

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore gentleMACS

Trascrizione

Riepilogo

Esplora Altri Video

Preparazione T fattore di crescita cellulare da splenociti Rat

La preparazione standardizzata di Single-Cell Sospensioni da tessuto polmonare del mouse utilizzando il dissociatore gentleMACS

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

L'uso di citometria a flusso per valutare lo stato della cromatina in cellule T

Isolamento delle cellule spermatogeniche murine utilizzando un colorante legante del DNA permeabile alle cellule violetto

Aggregazione cellulare incapsulata della matrice della membrana basale per lo studio della formazione del tessuto milza murino

Isolamento e trasferimento adottivo di sale alta trattati cellule dendritiche presentanti l'antigene

Caratterizzazione citometrica a flusso dello sviluppo delle cellule B murine

Isolamento e caratterizzazione di esosomi derivati da tessuti di milza di topo

Isolamento e caratterizzazione di esosomi derivati da tessuti di milza di topo