Die Autoren sind Mitarbeiter von Miltenyi Biotec GmbH, Deutschland.

Um Arbeit unter sterilen Bedingungen, ist es empfehlenswert, alle Schritte in einer Sterilbank durchgeführt. 1. Materialien <ol><li> HEPES-Puffer: 10 mM HEPES-NaOH pH 7,4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2, 1,8 mM CaCl 2) </li><li> Collagenase D Lösung: Collagenase D: 100 mg / mL (Collagenase D&gt; 0,15 U / mg), in HEPES-Puffer </li><li> DNase I-Lösung: 20.000 U / mL DNase I </li><li> PBS-Puffer bei pH 7,2 </li><li> PEB-Puffer: 1 Teil BSA-Stammlösung in 20 Teile autoMACS Mundspüllösung </li><li> Tissue: Lunge von 6 / 7 Woche alt erwachsenen weiblichen BALB / C Maus, frei von angrenzenden Organen abgeleitet. </li><li> Optional: CD11c MicroBeads, MACS MS Spalten, MACS Separator für die Isolierung von dendritischen Zellen aus der Maus Lunge Einzel-Zell-Suspension </li></ol> 2. Distanziert das Lungengewebe <ol><li> Das Gewebe spülen in eine Petrischale mit PBS auf die Erythrozyten zu entfernen. </li><li> Transfer von maximal 450 mg Lungengewebe zu einer gentleMACS C Tube mit 4,9 ml HEPES-Puffer. </li><li> 100 l Collagenase D-Lösung auf eine Endkonzentration von 2 mg / ml Collagenase D. </li><li> Add 10 l DNase I-Lösung für bis zu 150 mg Gewebe (Endkonzentration von 40 U / ml DNase I) oder 20 ul DNase I für 150-450 mg Lungengewebe (Endkonzentration von 80 U / ml DNase I). </li><li> Verschließen Sie die C Rohr und befestigen Sie es auf die gentleMACS Dissociator. Dann starten Sie das Programm &quot;m_lung_01&quot;. </li><li> Inkubieren für 30 min bei 37 ° C mit automatischer Rotation oder manuelles Drehen alle 5 min. </li><li> Return Probe auf die gentleMACS Dissociator und starten Sie das Programm &quot;m_lung_02&quot;. </li></ol> 3. Filtration <ol><li> Bereiten Sie eine 50-ml-Tube zum Sammeln filtrierten Zellen durch das Ersetzen der Kappe mit 70 um Maschenweite Zellsieb. </li><li> Sobald die zweite gentleMACS Programm endet in Abhängigkeit von der Verteilung des Probenmaterials in die Röhre, können Sie das Rohr kurz zentrifugieren, um das Probenmaterial am Boden des Röhrchens zu sammeln. </li><li> Entfernen Sie die Zellen durch das Septum verschlossenen Deckel des C Rohr mit einem geeigneten 1000 ul Pipettenspitze und sie auf die Zelle Sieb. </li><li> Waschen Sie die Zelle Sieb mit 5 ml HEPES-Puffer bei RT. </li><li> Zentrifugieren Sie die Zellen, um ein Pellet in einer 50 ml Tube bei 300xg für 10 min. </li><li> Saugen Sie den Überstand und Zellpellet in Ihrer gewünschten Volumen von PEB-Puffer. </li></ol> Teil 4: Repräsentative Ergebnisse: <img src="/files/ftp_upload/1266/1266fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Lung Dissoziation mit der gentleMACS ™ Dissociator führte in 92% lebensfähige Zellen. Tote Zellen wurden Fluoreszenz mit Propidiumiodid (PI) (;: keine PI-Färbung linken Dot-Plot rechten Dot-Plot) gefärbt. <img src="/files/ftp_upload/1266/1266fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Dissoziation von Maus Lungengewebe mit dem gentleMACS Dissociator Ergebnisse in einem hohen Prozentsatz lebensfähiger Leukozyten und Endothelzellen. Die abgeleiteten Einzel-Zellsuspensionen wurden mit CD45-PE und CD146-FITC oder CD31-FITC gefärbt und mittels Durchflusszytometrie analysiert. <img src="/files/ftp_upload/1266/1266figure2c.png" alt="Abbildung 2C" /> Abbildung 3. Single-Zellsuspension aus der Maus Lungengewebe abgeleitet wurde mit CD11c-FITC und Anti-mPDCA-1-APC Maus CD11c tief m-PDCA-1 + plasmazytoide DCs sowie CD11c hoch Zellen erkennen gefärbt. <img src="/files/ftp_upload/1266/1266fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Anreicherung von dendritischen Zellen mit CD11c MicroBeads, ein MiniMACS Separator und zwei MS Spalten.

<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 diesem Video stellen wir eine neue Methode für die Dissoziation von Maus Lungengewebe. Wir zeigen, dass eine Kombination von mechanischen und enzymatischen Behandlung von Lungengewebe zu einem hohen Prozentsatz lebensfähiger Leukozyten und Endothel-Zellen ergab. Insbesondere wurde die mechanische Zerstörung des Gewebes durch die gentleMACS Dissociator erreicht. Die gentleMACS C Tubes umfassen einen Rotor - Stator-System, das distanziert Gewebe auf sanfte Weise. Das Verfahren wird durch die Programm-Einstellungen d...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<td>CD11c MicroBeads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order No. 130-052-001</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx">http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx</a></td>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<td>CD146-FITC</td>
<td>&nbsp;</td>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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

Die Herstellung von ein-Zellsuspensionen aus den Geweben ist eine wichtige Voraussetzung für viele Experimente in Zellforschung. Der Prozess der Dissoziation ganzen Organen erfordert spezifische Parameter, um eine hohe Anzahl von lebensfähigen Zellen in reproduzierbarer Weise zu erhalten. Die gentleMACS Dissociator optimiert diese Aufgabe mit einem einfachen, praktischen Protokoll. Das Gerät enthält vorprogrammierte Einstellungen, die für die effiziente, aber schonende Dissoziation von einer Vielzahl von Gewebearten, darunter Mauslungen optimiert sind. In dieser Veröffentlichung wird die Nutzung des gentleMACS Dissociator auf das Lungengewebe von Mäusen abgeleitet wird demonstriert.

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

Distanziert Zellen aus bestimmten Gewebearten erfordert spezifische Parameter für die Gewebe-Agitation um ein hohes Volumen an lebensfähigen, kultivierbaren Zellen zu erhalten. Die Miltenyi gentleMACS dissociator optimiert diese Aufgabe mit einem einfachen, praktischen Protokoll. In dieser Veröffentlichung wird die Verwendung dieser Vorrichtung auf das Lungengewebe erklärt.

Standardisierte Erstellung von Einzel-Zellsuspensionen aus Maus Lungengewebe mit dem gentleMACS Dissociator

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

34.3K Aufrufe.  Miltenyi Biotec,GmbH.  中文 日本語 

Serie: 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.

Erstellung von Einzel-Zellsuspensionen aus Maus-Milz mit dem 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 ...

Forschung

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.

Generierung von Einzel-Zellsuspensionen aus Maus Nervengewebe

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.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

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.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

Isolierung von Zellkernen aus schockgefrorenem Lebergewebe für Einzelzell-Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

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.

Einzelzell-Suspensionspräparat aus Nil-Tilapia-Darm für die Einzelzell-Sequenzierung

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 ...

Halbautomatische Isolierung von weißem Fettgewebe der Maus

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.

Autor im Rampenlicht: Halbautomatische Isolierung der stromalen vaskulären Fraktion aus weißem Fettgewebe der Maus mit einem Gewebedissoziator  

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.

Halbautomatische Isolierung der stromalen vaskulären Fraktion aus murinem weißem Fettgewebe mit Hilfe eines Gewebedissoziators

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

Zweistufiger Zellverdau der Halsschlagader der Maus

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.

Autor im Rampenlicht: Dissoziation von vaskulärem Gewebe und Erforschung einzelzelliger Subcluster für eine zielgerichtete Therapie 

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

Herstellung einer Einzelzellsuspension aus den Halsschlagadern der Maus für die Einzelzellsequenzierung

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 ...