機器の紹介<ol><li>組織は、実行可能な単細胞懸濁液に紫のC管を使用して、穏やかに解離している。 </li><li> PBS / BSA EDTAバッファーは細胞を解離するために使用されます。バッファーは、EDTAを含む溶液をすすぎautoMACSの20部にBSAのストック溶液の1部を添加して調製される。 </li><li> MACSプレセパレーションフィルターは、解離組織サンプルが正常に行わMACS分離とその後の分析を促進する、すべての細胞塊がないかようにするために使用されます。 </li></ol>脾臓細胞を解離<ol><li>全体脾臓をgentleMACSのDissociatorを使用して脾細胞の単細胞懸濁液にdisscociatedされています。 </li><li>手順を開始するには、脾臓の質量に応じてバッファーの量とCチューブをロードする。若年成人女性のBALB / cマウスの健康な脾臓は80から120まで約10mgの重量を量る。この質量の1〜2脾臓は、緩衝液3mlを必要とする。感染症のある高齢の動物または動物から脾臓はしばしば大きく、バッファに検討する必要があります。バッファのボリュームは、それに応じてスケーリングする必要があります。を10mLの総バッファ量を超えないようにしてください。 </li><li>緩衝液3mlを含むCチューブに切除した脾臓を追加。 </li><li> Cチューブのキャップがタイトであることを確認して、上下のキャップでdissociatorにCチューブを取り付けます。 </li><li> dissociatorの電源を入れます。 1から2脾臓の場合は、プログラムのm_spleen_01を選択します。プログラムが完了するまで56秒かかります。脾臓の数が大きい場合は、プログラムm_spleen_04を使用して、このプログラムは、脾臓の組織の最大720 mgのために使用することができます。 </li><li>プログラムの終了後、ネイティブの脾臓の組織では、脾臓細胞懸濁液中に解離している。 </li><li>約30秒間室温で300xgで全細胞懸濁液をスピンダウン。これは、回転子と固定子は、C管の下部に収集されるすべてのセルから解放されることを保証します。 </li><li> 1000年マイクロリットルピペッターを使用してC管のキャップにセプタムシール開口部を通してサンプルを削除します。 </li></ol>濾過のステップ<ol><li>濾過工程を開始するには、プレセパレーションフィルターまたは30μmの孔径を持つセルストレーナーに解離組織を適用する。ストレーナーは1-2脾臓または2つ以上の脾臓では50 mLのチューブに15 mLのチューブを適合する必要があります。 </li><li>細胞懸濁液が通過してみましょう。その後、緩衝液5 mLを適用することにより、フィルターを洗う。 </li><li>今、脾臓細胞ペレットにすべての脾細胞を収集するために室温で10分間、300xgで細胞懸濁液でチューブを遠心する。 </li><li>今、我々は解離脾細胞のペレットを持っていることを、我々は、上清を吸引除去し、バッファの適切な量で細胞ペレットを再懸濁します必要があります。 </li></ol> gentleMACSのDissociatorを使用して、脾臓の解離は、MACSQuant Analyzerを使用してフローサイトメトリーによって実証されている実行可能な脾細胞の割合が高いが、得られます。死んだ細胞はヨウ化プロピジウムで染色されています。提供される例では、前方側方散乱ドットプロットでは、調製した脾細胞を示しています。

すべてのプロトコルとデータシートがご利用いただけます<a href="http://www.miltenyibiotec.com"&gt; http://www.miltenyibiotec.com。</a&gt;。

退屈でしばしば痛みを伴うマニュアルの解離を避けるために、gentleMACSのDissociatorは1つが制御され、再現可能な条件の下で非常に効率的に組織を解離することができます。組織解離の手順は、高い生存率と細胞と収量の細胞懸濁液に優しいです。 Cチューブとの組み合わせでgentleMACSのDissociatorは、様々な組織からの単細胞懸濁液の調製のために設計されています。楽器は、マウスの脾臓、肝臓、肺、または神経組織を解離するためにあらかじめ設定されたプログラムの数が装備されています。プログラムとプロトコルの範囲は連続して追加の組織を含むように拡張されます。

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<td>gentleMACS&#x2122; Starting Kit</td>
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<td>Miltenyi Biotec</td>
<td>130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
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<td>C Tubes</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec </td>
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<td><a href="http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx">http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx</a></td>
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<td>13 mL Tubes</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec </td>
<td>130-091-596</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx">http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx</a></td>
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<td>autoMACS&#x2122; Rinsing Solution </td>
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<td>Miltenyi Biotec </td>
<td>130-091-222</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx">http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx</a></td>
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<td>Miltenyi Biotec </td>
<td>130-091-376</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx">http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx</a></td>
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<td>Miltenyi Biotec </td>
<td>130-041-407</td>
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<td>MACSQuant Analyzer</td>
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<td>Miltenyi Biotec </td>
<td>130-092-197</td>
<td>&nbsp;</td>
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<td>Propidium Iodide Solution</td>
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<td>Miltenyi Biotec</td>
<td>130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
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preparation of single-cell suspensions from mouse spleen with the gentlemacs dissociator

単細胞懸濁液は、細胞分離、細胞解析と細胞培養の実験のための前提条件です。退屈でしばしば痛みを伴うマニュアルの解離を避けるためにgentleMACSのDissociatorは1つが制御され、再現可能な条件の下で非常に効率的に組織を解離することができます。 gentleMACSのDissociatorは、最適に、ユーザーの安全と時間節約と標準化を組み合わせ、マウスの脾臓を切り離すことができます。このビデオでは、gentleMACS Dissociator、機械的に生存細胞懸濁液を生成するために特殊なチューブを使用して組織を混乱させることができる自動化された卓上型の装置を使用してマウスの脾臓を解離させる方法について説明します。解離に続いて、脾臓を、ろ過、遠心分離し、さらにアプリケーション用に再懸濁する。

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

このビデオでは、マウス脾細胞の単細胞懸濁液を準備するためにgentleMACSのDissociatorを使用して自動化された組織の解離のためのシンプルな、時間の節約テクニックを説明します。

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 ...

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preparation-single-cell-suspensions-from-mouse-spleen-with-gentlemacs

Research

JoVE Journal

Biology

37.6K ビュー.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

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

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 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 ...

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 ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

シングルセルシーケンシングのためのナイルティラピア腸からのシングルセル懸濁液調製(英語)

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

マウス白脂肪組織の半自動単離

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

著者スポットライト:組織解離器を使用したマウス白脂肪組織からの間質血管画分の半自動分離  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

マウス頸動脈の2段階細胞消化

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

著者スポットライト:血管組織の解離と標的治療のための単一細胞サブクラスターの探索 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

シングルセルシーケンシングのためのマウス頸動脈からのシングルセル懸濁液の調製

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...