Introduction à l&#39;équipement <ol><li> Le tissu est légèrement dissocié, en utilisant le violet Tubes C dans viables unicellulaires suspensions. </li><li> Le tampon PBS / BSA EDTA est utilisé pour dissocier les cellules. Le tampon est préparé en ajoutant une partie de la solution stock de BSA à 20 parties de solution de rinçage autoMACS contenant de l&#39;EDTA. </li><li> Le MACS Pré-Séparation filtres sont utilisés pour que les échantillons de tissus dissociés sont libres de tous les amas cellulaires, la promotion réussie de séparation MACS et l&#39;analyse subséquente. </li></ol> Dissocier les cellules de la rate <ol><li> Rate entiers sont disscociated dans une seule cellule de suspensions de splénocytes utilisant le dissociateur gentleMACS. </li><li> Pour commencer la procédure, la charge du tube C avec un volume de tampon en fonction de la masse de la rate. Une rate saine d&#39;un adulte jeune femelle souris BALB / c pèse environ 80-120 mg. Aux 1 à 2 rates de cette masse besoin de 3 mL de tampon. Rates provenant d&#39;animaux âgés ou des animaux atteints d&#39;infections sont souvent plus grandes et doit être pesé dans la mémoire tampon. Les volumes de tampon doit être modulée en conséquence. Ne pas dépasser un volume tampon total de 10 ml. </li><li> Ajouter la rate disséqués pour le tube C contenant 3 ml de tampon. </li><li> Assurez-vous que le bouchon du tube C est serré et fixer le tube C de la dissociateur avec le bouchon vers le bas. </li><li> Mettez le dissociateur. Pour 1-2 rate, sélectionnez m_spleen_01 programme. Le programme va prendre 56 secondes pour terminer. Si le nombre des rates est supérieure, l&#39;utilisation du programme m_spleen_04, ce programme peut être utilisé pour un maximum de 720 mg de tissu splénique. </li><li> Après l&#39;achèvement du programme du tissu splénique natif est dissocié en une suspension de splénocytes. </li><li> Isoler la suspension de cellules entières au 300xg à température ambiante pendant environ 30 secondes. Cela garantira que le rotor et le stator sont libérés de toutes les cellules qui sont recueillis au fond du tube C. </li><li> Retirer l&#39;échantillon à travers le septum étanche ouverture dans le capuchon du tube C en utilisant un pipetteur 1000 microlitre. </li></ol> Étape de filtration <ol><li> Pour commencer l&#39;étape de filtration, appliquer le tissu dissocié de la pré-filtre de séparation ou une passoire cellule avec 30 um de taille des pores. Le tamis doit s&#39;adapter à un tube de 15 ml pendant 1-2 rate ou un tube de 50 ml pendant plus de 2 rates. </li><li> Laissez la suspension cellulaire passer à travers. Puis laver le filtre en appliquant 5 ml de tampon. </li><li> Maintenant, centrifuger le tube avec la suspension cellulaire à 300xg, à température ambiante pendant 10 minutes pour recueillir toutes les splénocytes dans une cellule de la rate à granulés. </li><li> Maintenant, que nous avons une pastille de splénocytes dissociés, nous devons aspirer le surnageant et remettre le culot cellulaire dans une quantité appropriée de mémoire tampon. </li></ol> La dissociation rate utilisant le dissociateur gentleMACS donnera un pourcentage élevé de splénocytes viable, ce qui est démontré par cytométrie en flux à l&#39;aide de l&#39;analyseur MACSQuant. Les cellules mortes sont colorées par de l&#39;iodure de propidium. Dans l&#39;exemple fourni, l&#39;intrigue avant du côté de dispersion point indique les splénocytes préparé.

Tous les protocoles et fiches techniques sont disponibles à<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

Pour éviter de dissociations manuel fastidieux et souvent douloureux, le dissociateur gentleMACS permet de dissocier les tissus de manière très efficace dans des conditions contrôlées et reproductibles. La procédure de dissociation des tissus est doux pour les cellules et les suspensions rendements cellulaires avec un taux de viabilité élevés. Le dissociateur gentleMACS en combinaison avec le C Tubes est conçu pour la préparation des suspensions unicellulaires à partir de différents tissus. L&#39;instrument est équipé d&#39;un certain nombre de programmes préréglés pour dissocier la rate de souris, le foie, les poumons ou les tissus nerveux. L&#39;éventail des programmes et des protocoles sera continuellement élargi pour inclure d&#39;autres tissus.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<td>autoMACS&#x2122; Rinsing Solution </td>
<td>&nbsp;</td>
<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>
</tr>
<tr>
<td>MACS&#174; BSA Stock Solution</td>
<td>&nbsp;</td>
<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>
</tr>
<tr>
<td>Pre-Separation Filters </td>
<td>&nbsp;</td>
<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>
</tr>
<tr>
<td>MACSQuant Analyzer</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec </td>
<td>130-092-197</td>
<td>&nbsp;</td>
<tr>
<td>Propidium Iodide Solution</td>
<td>&nbsp;</td>
<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

Une seule cellule suspensions sont un préalable pour des expériences dans la séparation des cellules, l&#39;analyse cellulaire et culture cellulaire. Pour éviter de dissociations manuel fastidieux et souvent douloureux de la dissociateur gentleMACS permet de dissocier les tissus de manière très efficace dans des conditions contrôlées et reproductibles. Le dissociateur gentleMACS peut dissocier de manière optimale la rate de souris, combinant un gain de temps et de normalisation par l&#39;utilisateur de sécurité. Cette vidéo décrit comment dissocier les rates de souris en utilisant le dissociateur gentleMACS, un système automatisé de paillasse dispositif qui peut perturber la mécanique des tissus en utilisant des tubes spéciaux pour produire des suspensions de cellules viables. Suite à la dissociation, les rates sont filtré, centrifugé et resuspendu pour d&#39;autres applications.

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

Cette vidéo décrit un simple gain de temps technique pour la dissociation des tissus automatisée en utilisant les dissociateur gentleMACS pour préparer une seule cellule de suspensions de splénocytes de souris.

Préparation des suspensions unicellulaires à partir de rate de souris avec le dissociateur gentleMACS

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 vues.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

Vidéo: 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.

Préparation standardisée des suspensions unicellulaires provenant de tissus pulmonaires de la souris en utilisant les dissociateur gentleMACS

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

Recherche

Biologie

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.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

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.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Technique simple et efficace pour la préparation de suspensions de cellules testiculaires

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.

Préparation de suspension unicellulaire de l’intestin de tilapia du Nil pour séquençage unicellulaire

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 ...

Isolement semi-automatisé de tissu adipeux blanc murin

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.

Pleins feux sur l’auteur : Isolement semi-automatisé de la fraction vasculaire stromale à partir de tissu adipeux blanc murin à l’aide d’un dissociateur tissulaire  

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.

Isolement semi-automatisé de la fraction vasculaire stromale à partir de tissu adipeux blanc murin à l’aide d’un dissociateur tissulaire

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

Digestion cellulaire en deux étapes de l’artère carotide de souris

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.

Pleins feux sur l’auteur : Dissociation du tissu vasculaire et exploration de sous-groupes unicellulaires pour une thérapie ciblée 

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

Préparation d’une suspension unicellulaire à partir d’artères carotides de souris pour le séquençage unicellulaire

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 ...