设备简介<ol><li>纸巾轻轻分离，使用紫彗星管成可行的单细胞悬液。 </li><li> PBS / BSA EDTA缓冲液是用来分解细胞。缓冲区是准备加入1 BSA的联合解决方案的冲洗液含EDTA autoMACS 20部分。 </li><li>互助委员会预分离过滤器是用来使分离的组织样本中的所有细胞团块，促进成功磁珠分离和后续分析。 </li></ol>游离的脾细胞<ol><li>全脾disscociated使用gentleMACS Dissociator脾单细胞悬液。 </li><li>要开始的程序，负载​​的缓冲液C管，根据脾脏的质量。一个健康的一个年轻的成年雌性BALB / c小鼠脾重约80-120毫克。这种大规模的每1至2脾脏需要3缓冲液。年龄较大的动物或感染动物的脾脏往往更大，必须在缓冲区权衡。缓冲区卷，必须相应地调整。不要超过10毫升缓冲液。 </li><li>添加解剖脾包含3毫升缓冲到C管。 </li><li>确保了C管帽紧，并重视与第C管dissociator下来。 </li><li>电源了dissociator。 1-2脾脏，选择方案m_spleen_01。该方案将需要56秒钟即可完成。如果脾脏数量较大，使用方案m_spleen_04;这个方案，可用于高达720毫克脾组织。 </li><li>程序完成后本机的脾组织分离成脾细胞悬液。 </li><li>在室温下约30秒300xg离心整个细胞悬液。这将确保转子和定子是在C管的底部收集的所有细胞中解脱出来。 </li><li>通过隔密封在C管帽，使用1000微升pipetter开放取出样品。 </li></ol>过滤步骤<ol><li>要开始过滤步骤，应用游离组织的预分离的过滤器或用30微米孔径的细胞滤网。超过2脾，过滤网应适合1-2脾脏或50毫升管15毫升管。 </li><li>让细胞悬浮液通过。然后申请5缓冲液冲洗过滤器。 </li><li>现在与细胞悬液，离心管在300xg在室温下，10分钟收集所有的脾细胞，脾细胞沉淀。 </li><li>现在，我们有一个沉淀分离脾，我们必须吸出上清液，重悬细胞沉淀在一个适当的缓冲区数额。 </li></ol>使用gentleMACS Dissociator脾虚分解会产生一个可行的脾细胞的比例很高，这是由流动使用MACSQuant分析仪流式细胞仪证明。死细胞染色，碘化丙啶。在所提供的例子，向前侧散射点图显示的准备脾。

可在所有的协议和数据表<a href="http://www.miltenyibiotec.com"&gt; http://www.miltenyibiotec.com。</a&gt;。

为了避免繁琐而且往往是痛苦的手动的脑机制分离，gentleMACS Dissociator允许游离于组织非常有效地控制和可重复的条件下。组织分解过程是温和的细胞与细胞悬浮液的产量高存活率。 gentleMACS Dissociator与C管结合的目的是准备从各种组织中的单细胞悬液。该仪器配备了预先设定的方案，以分解小鼠脾，肝，肺，或神经组织。方案和协议的范围将不断扩大，以包括额外的组织。

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<td>gentleMACS&#x2122; Starting Kit</td>
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<td>Miltenyi Biotec</td>
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<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>
<td>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>
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<td>13 mL Tubes</td>
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<td>Miltenyi Biotec </td>
<td>130-091-596</td>
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<td>autoMACS&#x2122; Rinsing Solution </td>
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<td>Miltenyi Biotec </td>
<td>130-091-222</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>
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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

单细胞悬液，细胞分离，细胞分析，细胞培养实验的一个先决条件。为了避免繁琐而且往往是痛苦的手动的脑机制分离gentleMACS Dissociator允许游离于组织非常有效地控制和可重复的条件下。 gentleMACS Dissociator最佳游离于小鼠脾，结合用户的安全省时和标准化。本视频介绍了如何使用gentleMACS Dissociator，一个自动化的台式设备，可使用特殊管产生可行的细胞悬浮液的机械破坏组织游离于小鼠脾脏。以下分解，脾脏被过滤，离心，并为进一步的应用重悬。

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

本视频介绍了一个简单的自动组织使用gentleMACS Dissociator准备小鼠脾单细胞悬液的解离，节省时间的技术。

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 ...

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

Research

JoVE Journal

Biology

37.5K 次观看.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

视频: 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.

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 ...

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 ...

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.

简单和高效的睾丸细胞悬液的制备技术

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.

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 ...