著者らは、ミルテニーバイオテク社、ドイツの従業員です。

無菌条件下で動作するように、それは層流フード内のすべての手順を実行することをお勧めします。 1。材料<ol><li> HEPES緩衝液：10mMのHEPES - NaOHでpH7.4の、150mMのNaCl、5mMのKClを、1mMのMgCl 2、1.8 mMのCaCl 2で ） </li><li>コラゲナーゼD溶液：コラゲナーゼD：HEPESバッファーで100 mg / mLの（コラゲナーゼD&gt; 0.15 U / mg）を、 </li><li> DNase I溶液：20,000 U / mlのDNase I </li><li> pH7.2のPBSバッファー</li><li> PEBバッファー：20部autoMACSすすぎ溶液の1部BSAストック溶液</li><li>組織：隣接臓器の自由な6月7日週齢の成体雌のBALB / Cマウス由来の肺。 </li><li>オプション：CD11cはマイクロビーズ、MACS MSカラム、マウスの肺単一細胞の懸濁液から樹状細胞の分離におけるMACSセパレーター</li></ol> 2。肺組織を解離<ol><li>赤血球を除去するためにPBSを含むペトリ皿の中で組織をすすいでください。 </li><li> 4.9 mLのHEPES緩衝液を含むgentleMACS Cチューブに450mgの肺組織の最大値を転送する。 </li><li> 2 mg / mlのコラゲナーゼDの最終濃度に100μLのコラゲナーゼD溶液を加える</li><li>最大150 mgの組織（40 U / mlのDNase Iの最終濃度）または150から450 mgの肺組織の場合は20μLのDNase I（80 U / mlのDNase Iの最終濃度）に10μLのDNase I溶液を加える。 </li><li>しっかりとCチューブを閉じ、gentleMACSのDissociatorにそれを添付してください。次にプログラム&quot;m_lung_01&quot;を実行してください。 </li><li> 37℃で30分間インキュベート℃で自動化された回転または手動ですべての5分を回すと。 </li><li> gentleMACSのDissociatorにサンプルを返すとプログラム&quot;m_lung_02&quot;を実行してください。 </li></ol> 3。ろ過<ol><li> 70μmのメッシュのセルストレーナーにキャップを交換することによってフィルタリングされた細胞を収集するための50 mlチューブを準備します。 </li><li> 1秒間に1回gentleMACSプログラムは、チューブ内の試料の分布に応じて、終了するには、チューブの下部にあるサンプルの材料を収集するために簡単にチューブを遠心することができる。 </li><li>適切な1000μlのピペットチップを使用してC管の隔膜密封キャップを介して細胞を取り出して、セルストレーナーにそれらを適用する。 </li><li>室温で5 mlのHEPES緩衝液でセルストレーナーを洗浄してください。 </li><li> 10分間300xgで50 mLのチューブにペレットに細胞を遠心分離します。 </li><li>上清を吸引除去し、PEBバッファーのご希望の音量に細胞ペレットを再懸濁します。 </li></ol>パート4：代表的な結果： <img src="/files/ftp_upload/1266/1266fig1.jpg" alt="図1" /> gentleMACS™Dissociatorと図1。肺解離は92％生存細胞をもたらした。死んだ細胞は蛍光ヨウ化プロピジウム（PI）（;：なしPI染色左のドットプロット右ドットプロット）で染色した。 <img src="/files/ftp_upload/1266/1266fig2.jpg" alt="図2" /> 図2。実行可能な白血球と内皮細胞の高い割合でgentleMACSのDissociatorの結果を使用してマウスの肺組織の解離。由来の単細胞懸濁液は、CD45 - PEとCD146 - FITCまたはCD31 - FITCで染色し、フローサイトメトリーによって分析した。 <img src="/files/ftp_upload/1266/1266figure2c.png" alt="図2C" /> マウスの肺組織由来の図3。単セル懸濁液をM - PDCA - 1マウスのCD11c 低を検出するために+形質樹だけでなく、CD11cは高い細胞をCD11cは- FITCおよび抗mPDCA - 1 - APCで染色した。 <img src="/files/ftp_upload/1266/1266fig4.jpg" alt="図4" /> 図4のCD11c MicroBeadsを用いて樹状細胞の濃縮、MiniMACSセパレータ二MSカラム。

<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.

このビデオでは、我々はマウスの肺組織の解離のための新しい方法を導入する。我々は、肺組織の機械的および酵素処理の組み合わせが可能な白血球と内皮細胞の高い割合をもたらしたことを示している。特に、組織の機械的な崩壊は、gentleMACSのDissociatorによって達成された。ステータシステム、穏やかな方法で解離の組織 - gentleMACS Cチューブは、ローターが含まれています。手順は、機器?...

<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

組織からの単細胞懸濁液の調製は、細胞の研究で多くの実験のための重要な前提条件です。全体の臓器を解離のプロセスは、再現可能な方法で生細胞の高い数値を得るために特定のパラメータが必要です。 gentleMACS Dissociatorは、シンプルで実用的なプロトコルを使用してこのタスクを最適化します。楽器は、マウスの肺を含む組織の種類、様々な効率的ですが穏やかな解離のために最適化され、事前にプログラム設定が含まれています。本書ではマウス由来の肺組織でgentleMACSのDissociatorの使用が示されている。

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

特定の組織型から解離し細胞は組織の攪拌のための特定のパラメータは、実行可能な、培養可能な細胞の大量を取得する必要があります。ミルテニーgentleMACS dissociatorは、シンプルで実用的なプロトコルを使用してこのタスクを最適化します。本書では肺組織でこの装置の使用は説明されています。

gentleMACS Dissociatorを使用してマウスの肺組織からの単細胞懸濁液の標準化の準備

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 ビュー.  Miltenyi Biotec,GmbH.  中文 日本語 

ビデオ: 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.

マウス肺樹状細胞の分離

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 ...

免疫・感染学

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とマウス脾臓からの単細胞懸濁液の調製

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.

マウス神経組織から単細胞懸濁液の生成

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.

離とマウスとヒトの肺胞マクロファージの体外培養

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.

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

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

シングルセル解析のための半自動機械酵素組織解離

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.

著者スポットライト:ドラッグデリバリーと免疫療法のためのナノ粒子技術の進歩

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.

電動装置を用いた単一細胞解析のための組織の機械的解離

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

生物工学

マウス肺における抗原特異的T細胞の単離と特性評価

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.

著者スポットライト:Optimized Protocol for Detecting Antigen-Specific T cells in Mouse Lungs Using Tetramers(テトラマーを用いたマウス肺の抗原特異的T細胞検出のための最適化されたプロトコール)

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.

ペプチド:主要組織適合遺伝子複合体四量体を用いたマウス肺由来の希少抗原特異的T細胞の同定

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

gentleMACS Dissociatorを使用してマウスの肺組織からの単細胞懸濁液の標準化の準備

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マウス肺樹状細胞の分離

gentleMACS Dissociatorとマウス脾臓からの単細胞懸濁液の調製

マウス神経組織から単細胞懸濁液の生成

離とマウスとヒトの肺胞マクロファージの体外培養

ヘキストの分離および特性評価^低 CD45^負マウス肺間葉系幹細胞

電動装置を用いた単一細胞解析のための組織の機械的解離

電動装置を用いた単一細胞解析のための組織の機械的解離

電動装置を用いた単一細胞解析のための組織の機械的解離

ペプチド:主要組織適合遺伝子複合体四量体を用いたマウス肺由来の希少抗原特異的T細胞の同定

ペプチド:主要組織適合遺伝子複合体四量体を用いたマウス肺由来の希少抗原特異的T細胞の同定