Name: preparation of single-cell suspensions from mouse spleen with the gentlemacs dissociator
Uploaded: 2008-12-11T00:00:00.000+00:00
Duration: 5 min 18 s
Description: Watch this Scientific Journal Video about Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator at JoVE.com

מבוא ציוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> רקמות הוא ניתק בעדינות, בעזרת צינורות סגול C לתוך תא בודד השעיות קיימא. </li><li style=";text-align:right;direction:rtl"> המאגר PBS / BSA EDTA משמש לנתק את התאים. המאגר מוכן ידי הוספת 1 חלק מהפתרון במלאי BSA עד 20 חלקים autoMACS שטיפת פתרון המכיל EDTA. </li><li style=";text-align:right;direction:rtl"> MACS טרום ההפרדה מסננים משמשים כך דגימות רקמה ניתק הן ללא כל גושים התא, לקדם הפרדה MACS מוצלח וניתוח שלאחר מכן. </li></ol> Dissociating בתאי הטחול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Spleens שלמות disscociated לתוך תא בודד השעיות של splenocytes באמצעות Dissociator gentleMACS. </li><li style=";text-align:right;direction:rtl"> כדי להתחיל את ההליך, לטעון את Tube C בהיקף של המאגר על פי המסה של spleens. הטחול בריא של עכבר צעיר מבוגר נשים BALB / c שוקל כ 80-120 מ&quot;ג. כל 1-2 spleens של מסה זו דורשת 3 מ&quot;ל של המאגר. Spleens מבעלי חיים או בבעלי חיים מבוגרים עם זיהומים הם לעתים קרובות גדולים יש לשקול במאגר. נפח המאגר חייב להיות מדורגים בהתאם. לא יעלה על נפח המאגר כולל של 10 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> מוסיפים את הטחול גזור אל התחתית C המכיל 3 מ&quot;ל של המאגר. </li><li style=";text-align:right;direction:rtl"> ודא כובע Tube C הוא חזק לצרף את Tube C כדי dissociator עם כובע ומטה. </li><li style=";text-align:right;direction:rtl"> כוח עד dissociator. במשך 1-2 spleens, בחר m_spleen_01 התוכנית. התוכנית ייקח 56 שניות כדי להשלים. אם מספר spleens גדול, להשתמש בתוכנית m_spleen_04: תוכנית זו יכולה לשמש עד 720 מ&quot;ג של רקמת טחול. </li><li style=";text-align:right;direction:rtl"> לאחר השלמת התוכנית רקמת טחול יליד הוא ניתק לתוך השעיה splenocyte. </li><li style=";text-align:right;direction:rtl"> ספין את ההשעיה התא כולו בבת 300xg בטמפרטורת החדר למשך כ -30 שניות. זה יבטיח כי הרוטור ואת הסטטור משוחררים של כל התאים אשר נאספים בתחתית של התחתית C. </li><li style=";text-align:right;direction:rtl"> הסר את המדגם דרך הפתח, מחצה חתום כובע Tube C באמצעות pipetter 1000 microliter. </li></ol> סינון צעד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להתחיל את שלב הסינון, להחיל את רקמת ניתק על סנן ההפרדה טרום או מסננת תא בגודל 30 מיקרומטר הנקבוביות. מסננת צריך להתאים צינור 15 מ&quot;ל במשך 1-2 spleens או שפופרת 50 מ&quot;ל במשך יותר מ 2 spleens. </li><li style=";text-align:right;direction:rtl"> בואו ההשעיה התא לעבור. ואז לשטוף את המסנן על ידי יישום של 5 מ&quot;ל חיץ. </li><li style=";text-align:right;direction:rtl"> עכשיו, צנטריפוגה הצינור עם ההשעיה התא 300xg, בטמפרטורת החדר למשך 10 דקות כדי לאסוף את כל splenocytes בתא הטחול גלולה. </li><li style=";text-align:right;direction:rtl"> עכשיו, שיש לנו גלולה של splenocytes ניתק, עלינו לשאוב supernatant ו resuspend התא גלולה בסכום המתאים של המאגר. </li></ol> דיסוציאציה הטחול באמצעות Dissociator gentleMACS תניב אחוז גבוה של splenocytes קיימא, אשר באה לידי ביטוי cytometry זרימה באמצעות Analyzer MACSQuant. תאים מתים הם מוכתמים על ידי יודיד propidium. בדוגמה סיפקה, לצד פיזור העלילה קדימה נקודה מציגה את splenocytes מוכן.

כל הפרוטוקולים וגליונות נתונים זמינים<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

כדי להימנע dissociations ידני מייגע וכואב לעיתים קרובות, את Dissociator gentleMACS מאפשרת לנתק רקמה ביעילות רבה בתנאים מבוקרים לשעתקו. ההליך רקמת ניתוק עדין לתאים התשואות השעיות תא עם קצב כדאיות גבוהה. Dissociator gentleMACS בשילוב עם C צינורות נועד להכנת תא בודד השעיות מרקמות שונות. המכשיר מצויד במספר שנקבע מראש תוכניות לנתק עכבר הטחול, הכבד, הריאות, או רקמה עצבית. מגוון של תוכניות ופרוטוקולים יורחב ברציפות לכלול רקמות נוספות.

<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

תא בודד המתלים הם תנאי הכרחי ניסויים ההפרדה התא, ניתוח התא תרבית תאים. כדי להימנע dissociations ידני מייגע וכואב לעיתים קרובות Dissociator gentleMACS מאפשרת לנתק רקמה ביעילות רבה בתנאים מבוקרים לשעתקו. Dissociator gentleMACS ניתן לנתק בצורה אופטימלית טחול עכבר, שילוב timesaving וסטנדרטיזציה עם בטיחות למשתמש. וידאו זה מתאר כיצד לנתק spleens העכבר באמצעות Dissociator gentleMACS, מכשיר אוטומטי הספסל העליון שיכולים לשבש באופן מכני לרקמות באמצעות צינורות מיוחדים כדי לייצר השעיות תא קיימא. בעקבות הניתוק, spleens מסוננים, centrifuged ו resuspended עבור יישומים נוספים.

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

וידאו זה מתאר פשוטים, חוסך זמן טכניקה ניתוק אוטומטי רקמות באמצעות Dissociator gentleMACS להכין תא בודד השעיות של splenocytes העכבר.

הכנת תא בודד השעיות מן הטחול עכבר עם 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

Universidad San Sebastian

Research

JoVE Journal

Biology

37.6K 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.

הכנת פקטור T צמיחת תאים מן Splenocytes עכברוש

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. ...

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.

הכנה סטנדרטית של תא בודד השעיות מתוך רקמת הריאה באמצעות עכבר 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.

הדור של תא בודד מרקמת השעיות עכבר עצבית

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

השימוש בcytometry הזרימה מנת להעריך את המצב של הכרומטין בתאי T

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

Immunology and Infection

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.

בידוד של תאי זרע מורין באמצעות צבע מחייב DNA סגול-נרגש תא חרוש

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

Developmental Biology

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.

מטריצת קרום מרתף צבירת תאים עטופה לחקר היווצרות רקמת טחול מורין

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.

בידוד והעברה המאמצת של מלח גבוהה מטופלים אנטיגן תאים דנדריטים

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.

אפיון ציטומטרי זרימה של פיתוח תאי מורין B

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>טכניקות זיקוק לבידוד אקסוזום המופק מטחול מורין

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>בידוד ואפיון אקסוזומים שמקורם ברקמת הטחול של העכבר

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

בידוד ואפיון אקסוזומים שמקורם ברקמת הטחול של העכבר

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

הכנת תא בודד השעיות מן הטחול עכבר עם Dissociator gentleMACS

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הכנת פקטור T צמיחת תאים מן Splenocytes עכברוש

הכנה סטנדרטית של תא בודד השעיות מתוך רקמת הריאה באמצעות עכבר Dissociator gentleMACS

הדור של תא בודד מרקמת השעיות עכבר עצבית

השימוש בcytometry הזרימה מנת להעריך את המצב של הכרומטין בתאי T

בידוד של תאי זרע מורין באמצעות צבע מחייב DNA סגול-נרגש תא חרוש

מטריצת קרום מרתף צבירת תאים עטופה לחקר היווצרות רקמת טחול מורין

בידוד והעברה המאמצת של מלח גבוהה מטופלים אנטיגן תאים דנדריטים

אפיון ציטומטרי זרימה של פיתוח תאי מורין B

בידוד ואפיון אקסוזומים שמקורם ברקמת הטחול של העכבר

בידוד ואפיון אקסוזומים שמקורם ברקמת הטחול של העכבר