Um Arbeit unter sterilen Bedingungen, ist es empfehlenswert, alle Schritte in einer Sterilbank durchgeführt. 1. Materialien <ul><li> Neural Gewebedissoziation Kit (P) (Komponenten: Lösung 1,2, 3 und 4, Storage Buffer) </li><li> Pre-Separation-Filter </li><li> (Optional) Myelin Removal Beads </li><li> Beta-Mercaptoethanol </li><li> gentleMACS Dissociator </li><li> C Tubes </li><li> MACSmix Tube Rotator </li><li> HBSS (w / o) </li><li> HBSS (w) </li></ul><ol><li> Stellen Sie folgende Lösungen vor Beginn des Protokolls: <ol><li> Hanks &#39;Salzlösung ohne zweiwertige Kationen Ca 2 + oder Mg 2 + (HBSS (w / o) </li><li> HBSS, Standard, dh mit Ca 2 + und Mg 2 + (HBSS (w) </li></ol></li><li> Abhängig von der Antigen-Epitop von Interesse in den folgenden Anwendungen verwenden entweder die Papain-basierte Neural Gewebedissoziation Kit (P) oder Trypsin-basierte Neural Gewebedissoziation Kit (T). </li><li> Bereiten Sie die folgenden Lösungen aus dem Nervengewebe Dissoziation Kit: <ol><li> Lösung 2: add beta-Mercaptoethanol bis 0,067 mM </li><li> Lösung 4: Das Pulver in 0,7 ml Lagerpuffer (sofern im Kit enthalten), vorsichtig mischen (nicht vortexen) </li><li> Enzyme Mix 1: Pipette 1,9 ml Lösung 2 und 50 ul Lösung 1 (beide aus Kit) in eine C Tube und Inkubation für 10-15 min bei 37 ° C. Das genügt, um 400 mg des Gehirngewebes zu distanzieren. </li></ol></li></ol> 2. Dissoziation der Nervengewebe <ol><li> Wiegen Hirngewebe in ein Röhrchen mit 1 ml HBSS (w / o) </li><li> Transfer-Maus Gehirn in der C Rohr mit 1950 ul vorgewärmtes Enzyme Mix 1. (Hinweis: Dieses Protokoll ist für ≤ 400 mg geschrieben, aber Lösung Volumes können skaliert auf bis zu 1600 mg Hirngewebe pro C Rohr sein) </li><li> Legen Sie die C Tube auf die gentleMACS Dissociator und Run-Programm &quot;m_brain_01&quot;. </li><li> Inkubieren mit Rotation (4 min mit einem MACSmix Tube Rotator) für 15 min bei 37 ° C. </li><li> Legen Sie die C Tube auf die gentleMACS Dissociator und Run-Programm &quot;m_brain_02&quot;. </li><li> Während der Dissoziation Schritt vorzubereiten 30 ul Enzym Mix 2 pro 400 mg Gewebe durch vorsichtiges Mischen von 20 ul der Lösung 3 und 10 ul Lösung 4. </li><li> Add Enzyme Mix 2 der C Rohr über das Septum-Dichtkappe und vorsichtig mischen, ohne Verwirbelungen. </li><li> Inkubieren Sie die C Tube mit Rotation für 10 min bei 37 ° C im Brutschrank. </li><li> Legen Sie die C Tube auf die gentleMACS Dissociator und Run-Programm &quot;m_brain_03&quot;. </li><li> Inkubieren Sie die C Tube mit Rotation für 10 min bei 37 ° C im Brutschrank. </li><li> Kurz zentrifugieren, um die Probe am Boden des Röhrchens zu sammeln. </li></ol> 3. Filtration <ol><li> Wählen Sie einen Filter groß genug, um den Durchgang zu den Zellen von Interesse erlauben. Zum Beispiel sind Purkinjezellen zu groß, um durch eine 30 pm Filter passieren, aber Prominin-1 + Zellen. </li><li> Verwenden Sie einen suitable1000 ul Pipette auf die Zellen zu entfernen bilden die C Rohr durch das Septum verschlossenen Deckel und es auf die Pre-Separation-Filter auf einem 15 ml-Collection-Tube. (Hinweis: Bei größeren Stichproben mit einem 50 ml-Collection-Tube). </li><li> Waschen Sie den Filter mit 10 ml HBSS (w). </li><li> Centrifuge die gefilterten Zellen bei 300 xg für 10 min bei RT. </li><li> Resuspendieren der Überstand in Ihrem Medium oder Puffer der Wahl für spätere Anwendungen. </li><li> So entfernen Sie Myelin Trümmer, verwenden Myelin Removal Perlen. </li></ol> 4. Repräsentatives Ergebnis: siehe Abbildungen 1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="Abb. 1" /> Abbildung. 1 Das Gehirn Dissoziation mit der gentleMACS Dissociator führte in 97% lebensfähige Zellen, wie die durchflusszytometrische Analyse zeigt. <img src="/files/ftp_upload/1267/1267fig2.png" alt="Abb. 2" /> Abbildung. 2 Light-Aufnahme von Neurosphäre Bildung nach magnetischer Zellsortierung mit Anti-Prominin-1 MicroBeads nach 7 Tagen Kultivierung in MACS ® NeuroMedium mit MACS Supplement B27 PLUS ergänzt. Die Zellen wurden aus CD1 Gehirn der Maus (P3) mit dem Neural Gewebedissoziation Kit (P) vorbereitet. <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="Abb. 3.5" /> Abbildung. 3 Myelin Trümmer in Einzel-Zellsuspensionen erheblich beeinträchtigt Zellisolation und das Entfernen von Myelin Trümmer von Myelin Removal Perlen steigert die Effizienz Zelle Trennungen. Für MACS Separations mit Anti-Prominin-1 MicroBeads wurde P22 Maushirn dissoziiert mit dem Neural Gewebedissoziation Kit (P). Zellen aus der Einzel-Zell-Suspension wurden entweder direkt für die Trennung verwendet werden, oder wurden eingereicht, um einer Verarmung Myelin mit Myelin Removal Perlen. Vergleicht man die Trennung von Proben ohne und mit Myelin Entfernung, zeigt, dass die Reinheit höher ist für Proben mit früheren Myelin Entfernung.

<ol>
	<li>Hardt, O., Scholz, C., K&#252;sters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons.">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li><li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>K&#246;rnyei, Z., G&#243;cza, E., R&#252;hl, R., Orsolits, B., V&#246;r&#246;s, E., Szab&#243;, B., V&#225;govits, B., Madar&#225;sz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis.">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li><li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases.">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li><li>Skog, J., W&#252;rdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li></ol>

Die Autoren sind Mitarbeiter von Miltenyi Biotec GmbH, Deutschland.

Die gentleMACS Dissociator erleichtert die standardisierte Erstellung von Einzel-Zellsuspensionen von Nervengewebe in einem geschlossenen System. Neural Gewebedissoziation Kits sind optimiert, um Antigen-Epitope für weitere Anwendungen wie Immunofärbungen und immunomagnetische Zellseparation 1-5 benötigt bewahren. In diesem Protokoll zeigen wir die schonende enzymatische Spaltung von Gehirn der Maus mit dem gentleMACS Dissociator und der Neural Gewebedissoziation Kit (P), was in 97% lebensfähige Zellen. 100 mg Nervengewebe Ausbeuten zwischen 5x10 6 und 1x10 7 Zellen, je nach dem Alter der Wirtsgewebe. Die gezielte Prominin-1 + Zellen wurden isoliert mit Anti-Prominin-1 MicroBeads und anschließend in Kultur genommen. Das Protokoll wird gezeigt werden, noch effektiver mit den zusätzlichen Einsatz von Myelin Removal Beads bei der Arbeit mit neuronalen Gewebe von Mäusen&gt; P7 abgeleitet.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.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>Neural Tissue Dissociation Kit (P)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-628</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx</a></td>
</tr>
<tr>
<td>Pre-Separation Filters</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 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>Anti-Prominin-1 MicroBeads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-333</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx">http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx</a></td>
</tr>
<tr>
<td>Anti-Prominin-1-APC</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-335</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx">http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx</a></td>
</tr>
<tr>
<td>Myelin Removal Beads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-094-544</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx">http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.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></td>
</tr>
<tr>
<td>Neural Tissue Dissociation Kit (T)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-231</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.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>		</tbody>
		</table>
		</div>

generation of single-cell suspensions from mouse neural tissue

Innerhalb des Nervensystems, haben Hunderte von neuronalen und glialen Zelltypen beschrieben worden. Jeder Zelltyp im Gehirn oder Rückenmark hat ein Repertoire von Zelloberflächen-Moleküle oder molekulare Determinanten, durch die sie identifiziert und charakterisiert werden können. Derzeit erfordern robuste Zelle Identifikation und Trenntechnik Single-Cell-Präparate zu erzeugen und gleichzeitig die Begrenzung Zelltod und die Zerstörung der charakteristischen Oberfläche Protein sein. Die gentleMACS Dissociator, wenn es in Kombination mit Trypsin oder Papain-basierte Dissoziation Kits verwendet wird, kann effektiv und schonend distanzieren Hirngewebe unter Beibehaltung Antigen-Epitope und die Begrenzung der Zellverlust. Standardisierte Erstellung von Einzel-Zell-Suspensionen erfolgt über C Rohre und optimiert, Preset gentleMACS Programme. Einmal erzeugt, Einzel-Zellsuspensionen mit monoklonalen Konjugate wie Anti-Prominin-1 MicroBeads, die neuralen Vorläuferzellen zu identifizieren, oder weiter gereinigt mit Myelin Removal Beads behandelt werden.

Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Distanziert Zellen aus bestimmten Gewebearten erfordert spezifische Parameter für Gewebe aggitation zu einem hohen Volumen von 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 nerual Gewebe erklärt.

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 ...

generation-of-single-cell-suspensions-from-mouse-neural-tissue

Research

JoVE Journal

Biology

22.3K Aufrufe.  Miltenyi Biotec,GmbH.  中文 日本語 

Serie: Generation of Single-Cell Suspensions from Mouse Neural Tissue

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

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.

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 ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

Beziehen High Quality-RNA aus Einzel-Zellpopulationen in menschlichen Postmortem Gehirngewebe

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

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 ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Erzeugung von menschlichen Nierentubuliden aus Gewebe und Urin

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

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 ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

Isolierung und Transkriptomanalyse pflanzlicher Zelltypen

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

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 ...