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

장비 소개 <ol><li> 조직은 가능한 단일 셀 suspensions에 보라색 C 튜브를 사용하여 부드럽게 dissociated 있습니다. </li><li> PBS / BSA EDTA (에틸렌 다이아 민 테트라 초산) 버퍼는 세포를 떼어 놓다하는 데 사용됩니다. 버퍼는 EDTA (에틸렌 다이아 민 테트라 초산)를 포함하는 솔루션을 Rinsing autoMACS 20 부분 BSA 주식 솔루션 1 부분을 추가하여 준비가되어 있습니다. </li><li> 맥 사전 분리 필터는 성공적인 맥 분리 및 후속 분석을 추진, dissociated 조직 샘플은 모든 세포 대단히 짧은 시간 무료 수 있도록하는 데 사용됩니다. </li></ol> 비장 세포를 Dissociating <ol><li> 전체 spleens은 gentleMACS의 Dissociator를 사용 splenocytes의 단일 셀 suspensions에 disscociated 있습니다. </li><li> 절차를 시작하려면 spleens의 질량에 따라 버퍼의 볼륨 C 튜브를로드합니다. 젊은 성인 여성의 BALB / C 마우스의 건강한 비장은 80-120에 대한 MG 무게. 이 질량의 모든 1-2 spleens 버퍼 3 ML이 필요합니다. 감염과 오래된 동물이나 동물에서 Spleens는 종종 큰되며 버퍼에 무게해야합니다. 버퍼 볼륨 따라 크기를 조정해야합니다. 10 ML 총 버퍼 량을 초과하지 마십시오. </li><li> 버퍼 3 ML를 포함하는 C 튜브로 해부하는 비장을 추가합니다. </li><li> C 튜브 뚜껑이 꽉 있는지 확인하고 아래 뚜껑으로 dissociator에 C 튜브를 연결합니다. </li><li> dissociator 최대 전력. 1-2 spleens 들어, 프로그램 m_spleen_01을 선택합니다. 이 프로그램은 완료 56초를 취할 것입니다. spleens의 수가 큰 경우, 프로그램 m_spleen_04을 사용하여,이 프로그램은 비장 조직의 720 밀리그램을 위해 사용할 수 있습니다. </li><li> 프로그램의 완성 후에 기본 비장 조직 splenocyte 현탁액에 dissociated 있습니다. </li><li> 약 30 초에 대한 상온에서 300xg에서 전체 세포 현탁액을 스핀. 이것은 로터와 스테 이터가 C 튜브의 하단에 수집된 모든 세포의 해제되어 있는지 확인합니다. </li><li> 1000 microliter의 pipetter를 사용하여 C 튜브 캡의 심장 - 봉인 개방을 통해 샘플을 제거합니다. </li></ol> 여과 단계 <ol><li> 여과 단계를 시작하려면 사전 분리 필터 30 μm의 기공 크기가 셀 스트레이너에 dissociated 조직을 적용합니다. 스트레이너는 2 개 이상 spleens 1-2 spleens 또는 50 ML 튜브에 대한 15 ML 튜브에 맞게해야합니다. </li><li> 세포 현탁액이 통과하자. 그렇다면 버퍼의 5 ML을 적용하여 필터를 세척. </li><li> 자, 비장 세포 펠렛의 모든 splenocytes를 수집하는 10 분 동안 상온에서 300xg에서 세포 현탁액으로 튜브를 원심 분리기. </li><li> 이제 우리는 dissociated splenocytes의 펠렛을 가지고, 우리는 뜨는을 기음과 버퍼의 적절한 금액 세포 펠렛을 resuspend한다. </li></ol> gentleMACS의 Dissociator를 사용하여 비장의 분리는 MACSQuant 분석기를 사용 유동세포계측법으로 증명 가능한 splenocytes의 높은 비율을 얻을 것입니다. 죽은 세포는 propidium 요오드화물의로 물들어있다. 제공된 예제에서는 앞으로 사이드 분산형 점 줄거리는 준비된 splenocytes를 보여줍니다.

모든 프로토콜 및 데이터 시트는에서 구할 수 있습니다<a href="http://www.miltenyibiotec.com"&gt; http://www.miltenyibiotec.com.</a&gt;.

지루하고 고통스러운 종종 수동 dissociations를 방지하려면 gentleMACS의 Dissociator 하나는 제어하고 재현성 조건 하에서 매우 효율적으로 조직을 떼어 놓다 수 있습니다. 조직 분리 절차는 높은 생존 속도로 세포 수율 세포 suspensions에 부드러운 있습니다. C 튜브와 함께 gentleMACS의 Dissociator 다양한 조직에서 단일 셀 suspensions의 준비를 위해 설계되었습니다. 악기는 마우스 비장, 간, 폐, 또는 신경 조직을 떼어 놓다로 사전 설정된 프로그램의 숫자를 갖추고 있습니다. 프로그램 및 프로토콜의 범위는 지속적으로 추가 조직을 포함하도록 확장됩니다.

<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

단일 셀 suspensions는 세포 분리, 세포 분석 및 세포 배양 실험에 꼭 필요합니다. 지루하고 고통스러운 종종 수동 dissociations을 피하기 위해 gentleMACS의 Dissociator 하나는 제어하고 재현성 조건 하에서 매우 효율적으로 조직을 떼어 놓다 수 있습니다. gentleMACS의 Dissociator는 최적의 사용자 안전을 절약해주는 및 표준화를 결합, 마우스 비장을 떼어 놓다 수 있습니다. 이 동영상은 gentleMACS Dissociator, 기계적으로 생균 suspensions을 생산하기 위해 특수 튜브를 사용하여 조직을 중단 수있는 자동화된 벤치 - 탑 장치를 사용 마우스 spleens을 떼어 놓다하는 방법에 대해 설명합니다. 분리 후, spleens는 여과 centrifuged, 추가적인 애플 리케이션을위한 resuspended 있습니다.

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

이 동영상은 마우스 splenocytes의 단일 셀 suspensions를 준비하는 gentleMACS의 Dissociator를 사용하여 자동 조직 분리에 대한 간단한 시간 절약 기법을 설명합니다.

gentleMACS Dissociator과 마우스 비장에서 단일 셀 Suspensions의 준비

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 조회수.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

비디오: 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.

랫 Splenocytes에서 T 세포 성장 인자 준비

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. ...

연구

생물학

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.

gentleMACS Dissociator를 사용하여 마우스 폐 조직에서 단일 셀 Suspensions의 표준화 준비

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.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

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

유동 세포 계측법의 사용은 T 세포의 염색질의 상태를 평가하기 위해

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

면역학 및 감염병학

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.

보라색 흥분 세포 투과성 DNA 결합 염료를 사용하여 뮤린 정자 세포의 격리

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

발생학

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.

쥐 비장 조직 형성을 조사하기 위한 Basement Membrane Matrix Encapsulated Cell Aggregation

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.

절연 및 높은 소금의 입양 전송 처리 수지상 세포를 항 원 제시

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.

뮤린 B 세포 발달의 유동 세포 측정 특성화

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>Murine Spleen-Derived Exosome 분리를 위한 정제 기법

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>Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

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

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

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

gentleMACS Dissociator과 마우스 비장에서 단일 셀 Suspensions의 준비

필기록

요약

더 많은 비디오 탐색

이 비디오의 챕터

랫 Splenocytes에서 T 세포 성장 인자 준비

gentleMACS Dissociator를 사용하여 마우스 폐 조직에서 단일 셀 Suspensions의 표준화 준비

마우스 신경 조직에서 단일 셀 Suspensions의 생성

유동 세포 계측법의 사용은 T 세포의 염색질의 상태를 평가하기 위해

보라색 흥분 세포 투과성 DNA 결합 염료를 사용하여 뮤린 정자 세포의 격리

쥐 비장 조직 형성을 조사하기 위한 Basement Membrane Matrix Encapsulated Cell Aggregation

절연 및 높은 소금의 입양 전송 처리 수지상 세포를 항 원 제시

뮤린 B 세포 발달의 유동 세포 측정 특성화

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)