장비 소개 <ol><li> 조직은 가능한 단일 셀 suspensions에 보라색 C 튜브를 사용하여 부드럽게 dissociated 있습니다. </li><li> PBS / BSA EDTA (에틸렌 다이아 민 테트라 초산) 버퍼는 세포를 떼어 놓다하는 데 사용됩니다. 버퍼는 EDTA (에틸렌 다이아 민 테트라 초산)를 포함하는 솔루션을 Rinsing autoMACS 20 부분 BSA 주식 솔루션 1 부분을 추가하여 준비가되어 있습니다. </li><li> 맥 사전 분리 필터는 성공적인 맥 분리 및 후속 분석을 추진, dissociated 조직 샘플은 모든 세포 대단히 짧은 시간 무료 수 있도록하는 데 사용됩니다. </li></ol> 비장 세포를 Dissociating <ol><li> 전체 spleens은 gentleMACS의 Dissociator를 사용 splenocytes의 단일 셀 suspensions에 disscociated 있습니다. </li><li> 절차를 시작하려면 spleens의 질량에 따라 버퍼의 볼륨 C 튜브를로드합니다. 젊은 성인 여성의 BALB / C 마우스의 건강한 비장은 80-120에 대한 MG 무게. 이 질량의 모든 1-2 spleens 버퍼 3 ML이 필요합니다. 감염과 오래된 동물이나 동물에서 Spleens는 종종 큰되며 버퍼에 무게해야합니다. 버퍼 볼륨 따라 크기를 조정해야합니다. 10 ML 총 버퍼 량을 초과하지 마십시오. </li><li> 버퍼 3 ML를 포함하는 C 튜브로 해부하는 비장을 추가합니다. </li><li> C 튜브 뚜껑이 꽉 있는지 확인하고 아래 뚜껑으로 dissociator에 C 튜브를 연결합니다. </li><li> dissociator 최대 전력. 1-2 spleens 들어, 프로그램 m_spleen_01을 선택합니다. 이 프로그램은 완료 56초를 취할 것입니다. spleens의 수가 큰 경우, 프로그램 m_spleen_04을 사용하여,이 프로그램은 비장 조직의 720 밀리그램을 위해 사용할 수 있습니다. </li><li> 프로그램의 완성 후에 기본 비장 조직 splenocyte 현탁액에 dissociated 있습니다. </li><li> 약 30 초에 대한 상온에서 300xg에서 전체 세포 현탁액을 스핀. 이것은 로터와 스테 이터가 C 튜브의 하단에 수집된 모든 세포의 해제되어 있는지 확인합니다. </li><li> 1000 microliter의 pipetter를 사용하여 C 튜브 캡의 심장 - 봉인 개방을 통해 샘플을 제거합니다. </li></ol> 여과 단계 <ol><li> 여과 단계를 시작하려면 사전 분리 필터 30 μm의 기공 크기가 셀 스트레이너에 dissociated 조직을 적용합니다. 스트레이너는 2 개 이상 spleens 1-2 spleens 또는 50 ML 튜브에 대한 15 ML 튜브에 맞게해야합니다. </li><li> 세포 현탁액이 통과하자. 그렇다면 버퍼의 5 ML을 적용하여 필터를 세척. </li><li> 자, 비장 세포 펠렛의 모든 splenocytes를 수집하는 10 분 동안 상온에서 300xg에서 세포 현탁액으로 튜브를 원심 분리기. </li><li> 이제 우리는 dissociated splenocytes의 펠렛을 가지고, 우리는 뜨는을 기음과 버퍼의 적절한 금액 세포 펠렛을 resuspend한다. </li></ol> gentleMACS의 Dissociator를 사용하여 비장의 분리는 MACSQuant 분석기를 사용 유동세포계측법으로 증명 가능한 splenocytes의 높은 비율을 얻을 것입니다. 죽은 세포는 propidium 요오드화물의로 물들어있다. 제공된 예제에서는 앞으로 사이드 분산형 점 줄거리는 준비된 splenocytes를 보여줍니다.

모든 프로토콜 및 데이터 시트는에서 구할 수 있습니다<a href="http://www.miltenyibiotec.com"&gt; http://www.miltenyibiotec.com.</a&gt;.

지루하고 고통스러운 종종 수동 dissociations를 방지하려면 gentleMACS의 Dissociator 하나는 제어하고 재현성 조건 하에서 매우 효율적으로 조직을 떼어 놓다 수 있습니다. 조직 분리 절차는 높은 생존 속도로 세포 수율 세포 suspensions에 부드러운 있습니다. C 튜브와 함께 gentleMACS의 Dissociator 다양한 조직에서 단일 셀 suspensions의 준비를 위해 설계되었습니다. 악기는 마우스 비장, 간, 폐, 또는 신경 조직을 떼어 놓다로 사전 설정된 프로그램의 숫자를 갖추고 있습니다. 프로그램 및 프로토콜의 범위는 지속적으로 추가 조직을 포함하도록 확장됩니다.

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<td>gentleMACS&#x2122; Starting Kit</td>
<td>&nbsp;</td>
<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>
<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>
<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>MACS&#174; BSA Stock Solution</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>Pre-Separation Filters </td>
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<td>Miltenyi Biotec </td>
<td>130-041-407</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx">http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx</a></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

단일 셀 suspensions는 세포 분리, 세포 분석 및 세포 배양 실험에 꼭 필요합니다. 지루하고 고통스러운 종종 수동 dissociations을 피하기 위해 gentleMACS의 Dissociator 하나는 제어하고 재현성 조건 하에서 매우 효율적으로 조직을 떼어 놓다 수 있습니다. gentleMACS의 Dissociator는 최적의 사용자 안전을 절약해주는 및 표준화를 결합, 마우스 비장을 떼어 놓다 수 있습니다. 이 동영상은 gentleMACS Dissociator, 기계적으로 생균 suspensions을 생산하기 위해 특수 튜브를 사용하여 조직을 중단 수있는 자동화된 벤치 - 탑 장치를 사용 마우스 spleens을 떼어 놓다하는 방법에 대해 설명합니다. 분리 후, spleens는 여과 centrifuged, 추가적인 애플 리케이션을위한 resuspended 있습니다.

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

이 동영상은 마우스 splenocytes의 단일 셀 suspensions를 준비하는 gentleMACS의 Dissociator를 사용하여 자동 조직 분리에 대한 간단한 시간 절약 기법을 설명합니다.

gentleMACS Dissociator과 마우스 비장에서 단일 셀 Suspensions의 준비

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 조회수.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

비디오: 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 Dissociator를 사용하여 마우스 폐 조직에서 단일 셀 Suspensions의 표준화 준비

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

연구

생물학

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.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

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.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

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.

유도 만능의 줄기 세포에서 파생 마우스의 생성

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.

고환 세포 현탁액의 준비를 위해 간단하고 효율적인 기술

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.

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 ...

murine white 지방 조직의 반자동 분리

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.

저자 스포트라이트: 조직 해리제를 사용하여 Murine White 지방 조직에서 기질 혈관 분획의 반자동 분리  

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.

조직 해리기를 사용하여 쥐 백색 지방 조직에서 기질 혈관 분획의 반자동 분리

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

마우스 경동맥의 2단계 세포 분해

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.

저자 스포트라이트: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

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

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 ...