Gli autori sono dipendenti di Miltenyi Biotec GmbH, Germania.

Per lavorare in condizioni sterili, si raccomanda di eseguire tutti i passaggi in una cappa a flusso laminare. 1. Materiale <ol><li> HEPES tampone: 10 mm HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1,8 mM CaCl 2) </li><li> Collagenasi soluzione D: D Collagenasi: 100 mg / ml (Collagenasi D&gt; 0,15 U / mg), in HEPES tampone </li><li> DNasi I soluzione: 20.000 U / mL DNasi I </li><li> Tampone PBS a pH 7,2 </li><li> PEB buffer: 1 parte di soluzione stock BSA in 20 parti autoMACS soluzione di risciacquo </li><li> Tessuto: polmone derivati ​​da 6 / 7 settimana di vita adulto femmina BALB / C del mouse, privo di organi adiacenti. </li><li> Opzionale: microsfere CD11c, MACS Colonne MS, separatore MACS per l&#39;isolamento di cellule dendritiche da polmone del mouse cella singola sospensione </li></ol> 2. Dissociare il tessuto polmonare <ol><li> Lavare il tessuto in una capsula di Petri contenente PBS per rimuovere eritrociti. </li><li> Trasferire un massimo di 450 mg di tessuto polmonare stecca gentleMACS C contenente 4,9 ml di tampone HEPES. </li><li> Aggiungere 100 ul soluzione Collagenasi D per una concentrazione finale di 2 mg / ml Collagenasi D. </li><li> Aggiungere 10 L DNasi soluzione che ho fino a 150 mg di tessuto (concentrazione finale di 40 U / ml DNasi I) o 20 l DNasi I per 150-450 mg di tessuto polmonare (concentrazione finale di 80 U / mL DNasi I). </li><li> Chiudere bene il tubo C e attaccarlo sul dissociatore gentleMACS. Quindi eseguire il programma &quot;m_lung_01&quot;. </li><li> Incubare per 30 minuti a 37 ° C con rotazione automatica o manuale trasformando ogni 5 minuti. </li><li> Ritorno del campione al dissociatore gentleMACS ed eseguire il programma &quot;m_lung_02&quot;. </li></ol> 3. Filtrazione <ol><li> Preparare una provetta da 50 ml per la raccolta di cellule filtrata sostituendo il tappo con un colino 70 celle mesh micron. </li><li> Una volta che il secondo programma gentleMACS si conclude, a seconda della distribuzione di materiale campione nel tubo, si può centrifugare la provetta brevemente per raccogliere il materiale del campione sul fondo della provetta. </li><li> Rimuovere le cellule attraverso il tappo sigillato setto della metropolitana C con una punta adatta 1000 microlitri pipetta e applicarli al filtro delle cellule. </li><li> Lavare il filtro delle cellule con 5 ml di tampone HEPES a temperatura ambiente. </li><li> Centrifugare le cellule a una pallina in un tubo da 50 ml a 300xg per 10 minuti. </li><li> Aspirare il surnatante e risospendere il pellet di cellule nel volume desiderato di tampone PEB. </li></ol> Parte 4: Risultati Rappresentante: <img src="/files/ftp_upload/1266/1266fig1.jpg" alt="Figura 1" /> Figura 1. Polmone dissociazione con l&#39;™ gentleMACS dissociatore portato a 92% cellule vitali. Le cellule morte sono state fluorescenti colorate con ioduro di propidio (PI) (dot plot a destra; dot plot a sinistra: nessuna colorazione PI). <img src="/files/ftp_upload/1266/1266fig2.jpg" alt="Figura 2" /> Figura 2. Dissociazione del tessuto polmonare del mouse utilizzando i risultati dissociatore gentleMACS in un&#39;alta percentuale di leucociti vitali e cellule endoteliali. Il derivato cella singola sospensioni sono state colorate con CD45-PE e CD146-CD31-FITC o FITC e analizzate mediante citometria a flusso. <img src="/files/ftp_upload/1266/1266figure2c.png" alt="Figura 2C" /> Figura 3. Singola cellula sospensioni derivate da tessuti polmonari del mouse era macchiata di CD11c-FITC e anti-mPDCA-1-APC per rilevare il mouse CD11c bassa m-PDCA-1 + DC plasmacitoidi così come le cellule CD11c alto. <img src="/files/ftp_upload/1266/1266fig4.jpg" alt="Figura 4" /> Figura 4. Arricchimento delle cellule dendritiche con microsfere CD11c, un separatore MiniMACS e due colonne MS.

<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.

In questo video, si introduce un nuovo metodo per la dissociazione del tessuto polmonare mouse. Abbiamo dimostrato che una combinazione di trattamento meccanico ed enzimatica del tessuto polmonare ha prodotto un alta percentuale di leucociti vitali e cellule endoteliali. In particolare, la disintegrazione meccanica del tessuto è stato ottenuto il dissociatore gentleMACS. I tubi C gentleMACS includono un rotore - sistema dello statore, che il tessuto si dissocia in modo gentile. La procedura è controllata dalle imposta...

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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>
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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>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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<td>CD146-FITC</td>
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<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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<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

La preparazione di singole cellule dai tessuti sospensioni è un presupposto importante per molti esperimenti nel campo della ricerca cellulare. Il processo di dissociazione organi interi richiede specifici parametri per ottenere un elevato numero di cellule vitali in modo riproducibile. Il dissociatore gentleMACS ottimizza questa operazione con un semplice protocollo pratico. Lo strumento contiene le impostazioni pre-programmate che sono ottimizzati per la dissociazione efficiente ma gentile di una varietà di tipi di tessuto, tra cui i polmoni del mouse. In questa pubblicazione l&#39;utilizzo del dissociatore gentleMACS sul tessuto polmonare derivati ​​da topi è dimostrata.

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

Dissociare cellule di tipi di tessuto specifico richiede parametri specifici per l&#39;agitazione dei tessuti per ottenere un elevato volume di vitali, cellule coltivabili. Il Miltenyi gentleMACS dissociatore ottimizza questa operazione con un semplice protocollo pratico. In questa pubblicazione l&#39;utilizzo di questo apparecchio sul tessuto polmonare viene spiegata.

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 ...

standardized-preparation-single-cell-suspensions-from-mouse-lung

Research

JoVE Journal

Biology

34.4K Views.  Miltenyi Biotec,GmbH.  中文 日本語 

Video: 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.

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 ...

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 ...