作者是德国美天旎生物技术公司，雇员。

若要在无菌条件下，它是建议执行层流罩中的所有步骤。 1。材料<ol><li> HEPES缓冲液：10毫米HEPES - NaOH溶液pH值7.4，150 mM氯化钠，氯化钾5毫米，1毫米MgCl 2的 ，1.8毫米氯化钙2） </li><li>胶原酶D解决方案：胶原酶D：100毫克/毫升（胶原酶ð&gt; 0.15 U /毫克），在HEPES缓冲</li><li> DNA酶我的解决方案：2万U / mL的DNA酶我</li><li>在pH 7.2的PBS缓冲液</li><li> PEB的缓冲区：BSA在20部分第1部分股票解决方案autoMACS冲洗液</li><li>组织：从6 / 7周龄成年雌性BALB / C鼠，毗邻器官的肺。 </li><li>可选：表面CD11c微珠，MACS MS色谱柱，磁珠隔离从小鼠肺癌的单细胞悬液的树突状细胞的分离器</li></ol> 2。游离的肺组织<ol><li>一个Petri菜含有PBS去除红细胞冲洗组织。 </li><li>最大为450 mg肺组织转移到一个gentleMACS C管含有4.9 mL的HEPES缓冲。 </li><li>加入100μL的胶原酶D溶液的终浓度为2 mg / mL胶原酶D. </li><li>加入10μL的DNA酶高达150毫克的组织（终浓度为40 U / ml的DNA酶我）或20μL的DNA酶我为150-450毫克肺组织（终浓度为80 U / mL的DNA酶我）我的解决方案。 </li><li>紧紧贴着C管和附加到gentleMACS Dissociator它。然后运行该程序“m_lung_01”。 </li><li>孵育30分钟，在37 ° C，自动旋转或手动转动，每5分钟。 </li><li>返回样本gentleMACS Dissociator和运行方案“m_lung_02”。 </li></ol> 3。过滤<ol><li>准备取代第70微米网状细胞过滤器收集筛选细胞50毫升管。 </li><li>第二gentleMACS计划一旦结束，根据分布在管内的样品材料，您可能离心管简单地收集在试管底部的样品材料。 </li><li>通过隔膜密封的C管，使用第一个合适的1000μL枪头取出细胞，并将其应用到细胞过滤器。 </li><li> 5毫升HEPES缓冲液在室温下的细胞滤网清洗。 </li><li>一个颗粒细胞在300xg离心10分钟，在50毫升管。 </li><li>吸上清，重悬细胞沉淀在你想要的PEB的缓冲量。 </li></ol>第4部分：代表性的成果： <img src="/files/ftp_upload/1266/1266fig1.jpg" alt="图1" /> 图1。龙gentleMACS™Dissociator分解导致92％的活细胞。死细胞荧光染色，碘化丙啶（PI）（右点图，左点图：没有PI染色）。 <img src="/files/ftp_upload/1266/1266fig2.jpg" alt="图2" /> 图2。使用gentleMACS Dissociator结果在一个可行的白细胞和内皮细胞的高比例对小鼠肺组织分离。派生的单细胞悬液与CD45 - PE，CD146 - FITC或CD31 - FITC染色和流式细胞仪分析。 <img src="/files/ftp_upload/1266/1266figure2c.png" alt="图2C" /> 表面CD11c - FITC和反mPDCA - 1 - APC检测鼠标的CD11c 低 M - PDCA - 1 +浆区议会，以及表面CD11c 高细胞染色图3。小鼠肺组织所得的单细胞悬液。 <img src="/files/ftp_upload/1266/1266fig4.jpg" alt="图4" /> 图4。使用树突状细胞表面CD11c微珠，MiniMACS分离器和两个MS色谱柱的富集。

<ol>
	<li>Swanson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3-Ligand,+IL-4,+GM-CSF,+and+Adherence-Mediated+Isolation+of+Murine+Lung+Dendritic+Cells:+Assessment+of+Isolation+Technique+on+Phenotype+and+Function.">Flt3-Ligand, IL-4, GM-CSF, and Adherence-Mediated Isolation of Murine Lung Dendritic Cells: Assessment of Isolation Technique on Phenotype and Function.</a> J. Immunol. 173, 4875-4881 (2004).</li><li>Vermaelen, K., Pauwels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+simple+discrimination+of+mouse+pulmonary+dendritic+cell+and+macrophage+populations+by+flow+cytometry:+Methodology+and+new+insights.">Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: Methodology and new insights.</a> Cytometry Part A. 61A, 170-177 (2004).</li></ol>

The authors are employees of Miltenyi Biotec GmbH who make the instrument used in this article.

在这个视频中，我们引入一个新的小鼠肺组织的分离方法。我们展示的机械和酶治疗肺组织结合，取得了一个可行的白细胞和内皮细胞的高比例。具体来说，组织机械解体取得gentleMACS Dissociator。 gentleMACS彗星管包括一个转子 - 定子系统，它游离在一种温和的方式组织。过程控制仪器的程序设置。这些设置进行了优化，实现高产和细胞活力。派生的单细胞悬液与CD45 - PE，CD146 - FITC或CD31 - FITC染色和?...

<table><tbody><tr><td>C Tubes</td><td>Miltenyi Biotec</td><td>Order no. 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>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>Order no. 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>MACSmix&amp;#8482; Tube Rotator</td><td>Miltenyi Biotec</td><td>Order no. 130-090-753</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&lt;/a&gt;</td></tr><tr><td>autoMACS&amp;#8482; Rinsing Solution</td><td>Miltenyi Biotec</td><td>Order no. 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>Order no. 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>CD11c MicroBeads</td><td>Miltenyi Biotec</td><td>Order No. 130-052-001</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx&lt;/a&gt;</td></tr><tr><td>CD11c-FITC, CD11c-PE</td><td>Miltenyi Biotec</td><td>Order no. 130-092-026130-091-830</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx&lt;/a&gt;</td></tr><tr><td>Anti-mPDCA-1-APC</td><td>Miltenyi Biotec</td><td>Order no. 130-091-963</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx&lt;/a&gt;</td></tr><tr><td>CD146-FITC</td><td>Miltenyi Biotec</td><td>Order no. 130-092-026</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx&lt;/a&gt;</td></tr><tr><td>CD45-PE</td><td>Miltenyi Biotec</td><td>Order no. 130-091-610</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx&lt;/a&gt;</td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>Order no. 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><tr><td>Art 1000 Reach Pipet Tips</td><td>Molecular BioProducts</td><td>Order no. 2079</td><td>&lt;a href=&quot;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&quot;&gt;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.mbpinc.com/html/products/art/display.html&quot;&gt;http://www.mbpinc.com/html/products/art/display.html&lt;/a&gt;</td></tr></tbody></table>

standardized preparation of single-cell suspensions from mouse lung tissue using the gentlemacs dissociator

从组织单细胞悬液的制备细胞研究中的许多实验的重要前提。游离整个器官的过程中，需要在特定的参数，在重现的方式获取大量的活菌。 gentleMACS Dissociator优化这个任务简单，实用的协议。该仪器包含预编程的优化设置的多种组织类型，包括小鼠肺的效率，但温和的分解。在本出版物中使用从小鼠的肺组织gentleMACS Dissociator证明。

Watch this Scientific Journal Video about Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator at JoVE.com

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

游离特定的组织类型的细胞，组织鼓动的具体参数，以获得一个可行的，可培养细胞的高容量。美天旎gentleMACS dissociator优化这个任务简单，实用的协议。在本出版物中使用本仪器对肺组织进行了解释。

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

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

standardized-preparation-single-cell-suspensions-from-mouse-lung

Research

JoVE Journal

Biology

34.5K 次观看.  Miltenyi Biotec,GmbH.  中文 日本語 

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

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

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

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

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

生物学

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.

从小鼠神经组织产生单细胞悬浮液

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

用于单细胞分析的半自动机械酶组织解离

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. Manual protocols for single-cell isolations are highly time-consuming and prone to user variability. Automated mechanical protocols are able to reduce processing time and sample variability but aren't easily accessible or cost-effective in lower-resourced research settings. The device described here was designed for semi-automated tissue dissociation using commercially available materials as a low-cost alternative for academic laboratories. Instructions to fabricate, assemble, and operate the device design have been provided. The dissociation protocol reliably produces single-cell suspensions with comparable cell yields and sample viability to manual preparations across multiple mouse tissues. The protocol provides the ability to process up to 12 tissue samples simultaneously per device, making studies requiring large sample sizes more manageable. The accompanying software also allows for customization of the device protocol to accommodate varying tissues and experimental constraints.

作者聚焦：用于药物递送和免疫治疗的纳米颗粒技术的进展

A general protocol for the combined enzymatic and semi-automated mechanical dissociation of tissues to generate single-cell suspensions for downstream analyses, such as flow cytometry, is provided. Instructions for the fabrication, assembly, and operation of the low-cost mechanical device developed for this protocol are included.

使用电动设备对组织进行机械解离以进行单细胞分析

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. ...

生物工程

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

副本

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此视频中的章节

小鼠肺树突状细胞的分离

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

从小鼠神经组织产生单细胞悬浮液

小鼠与人肺泡巨噬细胞的分离和体外培养

对赫斯特的分离与表征^低 CD45^负小鼠肺间质干细胞

使用电动设备对组织进行机械解离以进行单细胞分析

使用电动设备对组织进行机械解离以进行单细胞分析

使用电动设备对组织进行机械解离以进行单细胞分析

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞