Introduzione alla Attrezzature <ol><li> Il tessuto è leggermente dissociato, utilizzando viola Tubi C in singola cellula vitale sospensioni. </li><li> La PBS / BSA tampone EDTA è usato per separare le cellule. Il buffer è preparato con l&#39;aggiunta di 1 parte di soluzione madre BSA per 20 parti di autoMACS Risciacquo Soluzione contenente EDTA. </li><li> La pre-separazione filtri MACS sono usati in modo che i campioni di tessuto dissociate sono liberi di tutti i grumi di cellule, favorendo il successo separazione MACS e successiva analisi. </li></ol> Dissociare le cellule della milza <ol><li> Milze intere disscociated in cella singola sospensioni di splenociti utilizzando il dissociatore gentleMACS. </li><li> Per iniziare la procedura, caricare la metropolitana C, con un volume di tampone a seconda della massa della milza. Una milza sana di un giovane adulto femmina BALB / c del mouse pesa circa 80-120 mg. Ogni 1 o 2 milza di questa massa richiedono 3 ml di tampone. Milza di animali più vecchi o animali con infezioni sono spesso più grandi e devono essere valutati nel buffer. I volumi buffer deve essere ripartita in proporzione. Non superano un volume totale di buffer di 10 ml. </li><li> Aggiungere la milza sezionato per la metropolitana C contenente 3 ml di buffer. </li><li> Assicurarsi che il tappo del tubo C è stretto e collegare il tubo C al dissociatore con il tappo verso il basso. </li><li> Accendere il dissociatore. Per 1-2 milza, selezionare m_spleen_01 programma. Il programma avrà 56 secondi per essere completata. Se il numero di milza è maggiore, usare il programma m_spleen_04, questo programma può essere utilizzato per un massimo di 720 mg di tessuto milza. </li><li> Dopo il completamento del programma del tessuto milza nativa è dissociato in una sospensione splenocyte. </li><li> Spin down la sospensione a cellule intere, a 300xg a temperatura ambiente per circa 30 sec. Questo farà sì che il rotore e lo statore sono liberati di tutte le cellule che vengono raccolte sul fondo del tubo C. </li><li> Rimuovere il campione attraverso il setto-sigillato l&#39;apertura del tappo del tubo C con una 1000 pipetter microlitro. </li></ol> Filtrazione passo <ol><li> Per iniziare la fase di filtrazione, applicare il tessuto dissociato al pre-filtro di separazione o di un colino cellula con 30 micron dimensioni dei pori. Il filtro dovrebbe montare un tubo di 15 ml per 1-2 milza o un tubo da 50 ml per più di 2 milze. </li><li> Lasciate che la sospensione di cellule passano attraverso. Poi lavare il filtro applicando 5 ml di tampone. </li><li> Ora, centrifugare la provetta con la sospensione cellulare a 300xg, a temperatura ambiente per 10 minuti per raccogliere tutte splenociti in una milza pellet. </li><li> Ora, che abbiamo un pellet di splenociti dissociato, dobbiamo aspirare il supernatante e risospendere il pellet cellulare in una quantità adeguata di buffer. </li></ol> Dissociazione milza utilizzando il dissociatore gentleMACS produrrà una elevata percentuale di splenociti vitale, che è dimostrato mediante citometria di flusso utilizzando l&#39;analizzatore MACSQuant. Le cellule morte sono colorate con ioduro di propidio. Nell&#39;esempio fornito, l&#39;attaccante scatter plot mostra il lato dot splenociti preparato.

Tutti i protocolli e le schede tecniche sono disponibili all&#39;indirizzo<a href="http://www.miltenyibiotec.com"&gt; Http://www.miltenyibiotec.com.</a&gt;.

Per evitare di dissociazioni manuali ripetitive e spesso dolorosa, il dissociatore gentleMACS permette di dissociare i tessuti in maniera molto efficiente, in condizioni controllate e riproducibili. La procedura di dissociazione tessuto è delicato per le cellule e sospensioni cellulari produce un alto tasso di vitalità. Il dissociatore gentleMACS in combinazione con C Tubi è stata progettata per la preparazione di singola cellula sospensioni da vari tessuti. Lo strumento è dotato di una serie di programmi preimpostati per dissociare il mouse milza, fegato, polmone, o tessuto neurale. La gamma di programmi e protocolli sarà continuamente ampliata per includere altri tessuti.

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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>
</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>
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<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>
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<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>
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<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>
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<td>MACSQuant Analyzer</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec </td>
<td>130-092-197</td>
<td>&nbsp;</td>
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<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

Monocellulari sospensioni sono un prerequisito per esperimenti di separazione delle cellule, l&#39;analisi delle cellule e coltura delle cellule. Per evitare di dissociazioni manuali ripetitive e spesso doloroso il dissociatore gentleMACS permette di dissociare i tessuti in maniera molto efficiente, in condizioni controllate e riproducibili. Il dissociatore gentleMACS può dissociarsi in modo ottimale milza del mouse, coniugando risparmio di tempo e di normalizzazione dall&#39;utente sicurezza. Questo video spiega come dissociare milza del mouse utilizzando il dissociatore gentleMACS, un sistema automatico da banco dispositivo in grado di distruggere meccanicamente i tessuti con tubi speciali per la produzione di sospensioni di cellule vitali. A seguito di dissociazione, milza vengono filtrati, centrifugati e risospesi per ulteriori applicazioni.

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

Questo video descrive un semplice, di risparmiare tempo tecnica per la dissociazione dei tessuti automatizzato mediante il dissociatore gentleMACS per preparare cella singola sospensioni di splenociti mouse.

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore 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 Views.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

Video: 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.

La preparazione standardizzata di Single-Cell Sospensioni da tessuto polmonare del mouse utilizzando il dissociatore gentleMACS

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

Ricerca

Biologia

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.

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

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.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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.

Tecnica semplice ed efficace per la preparazione delle sospensioni cellulari testicolari

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.

Preparazione della sospensione unicellulare dall'intestino della tilapia del Nilo per il sequenziamento a singola cellula

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 ...

Isolamento semi-automatico del tessuto adiposo bianco murino

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.

Autore in primo piano: Isolamento semi-automatico della frazione vascolare stromale dal tessuto adiposo bianco murino utilizzando un dissociatore tissutale  

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.

Isolamento semi-automatico della frazione vascolare stromale dal tessuto adiposo bianco murino mediante un dissociatore tissutale

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

Digestione cellulare in due fasi dell'arteria carotide di topo

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.

Autore in primo piano: Dissociazione del tessuto vascolare ed esplorazione di sottocluster di singole cellule per la terapia mirata 

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

Preparazione di una sospensione unicellulare da arterie carotidi di topo per il sequenziamento di singole cellule

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 ...