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?...

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<tr>
<td>C Tubes</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-237</td>
<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>
</tr>
<tr>
<td>gentleMACS&#x2122; Starting Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 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>
</tr>
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<td>MACSmix&#x2122; Tube Rotator</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
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<td>autoMACS&#x2122; Rinsing Solution</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 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>MACS&#174; BSA Stock Solution</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 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>CD11c MicroBeads</td>
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<td>Miltenyi Biotec</td>
<td>Order No. 130-052-001</td>
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<td>CD11c-FITC, CD11c-PE</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-026	130-091-830</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx">http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx</a></td>
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<td>Anti-mPDCA-1-APC</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-091-963</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx">http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx</a></td>
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<tr>
<td>CD146-FITC</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-026</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx">http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx</a></td>
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<td>CD45-PE</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-091-610</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx">http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx</a></td>
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<td>Propidium Iodide Solution</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 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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<tr>
<td>Art 1000 Reach Pipet Tips</td>
<td>&nbsp;</td>
<td>Molecular BioProducts</td>
<td>Order no. 2079</td>
<td><a href="http://www.mbpinc.com/ASP/products.aspx?query_TipID=61">http://www.mbpinc.com/ASP/products.aspx?query_TipID=61</a> <a href="http://www.mbpinc.com/html/products/art/display.html">http://www.mbpinc.com/html/products/art/display.html</a>
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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

34.3K 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

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 ...

科研

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 ...

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.

单从小鼠锥感光细胞吸录音

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.

诱导多能干细胞来源于小鼠的生成

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

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

从快速冷冻肝组织中分离细胞核用于单细胞多组学

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

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.

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

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 ...

小鼠白色脂肪组织的半自动分离

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.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

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.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

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

小鼠颈动脉的两步细胞消化

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.

作者聚焦：血管组织解离和探索用于靶向治疗的单细胞亚簇 

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

从小鼠颈动脉制备用于单细胞测序的单细胞悬液

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 ...