Per lavorare in condizioni sterili, si raccomanda di eseguire tutti i passaggi in una cappa a flusso laminare. 1. Materiale <ul><li> Neurali del tessuto dissociazione Kit (P) (Componenti: Soluzione 1,2, 3 e 4, buffer di memoria) </li><li> Pre-filtri di separazione </li><li> (Opzionale) Mielina rimozione Beads </li><li> Beta-mercaptoetanolo </li><li> gentleMACS dissociatore </li><li> C Tubi </li><li> MACSmix tubo Rotator </li><li> HBSS (w / o) </li><li> HBSS (w) </li></ul><ol><li> Preparare le seguenti soluzioni prima di iniziare la procedura: <ol><li> Sale Hanks &#39;Soluzione tampone senza cationi bivalenti Ca 2 + o Mg 2 + (HBSS (w / o) </li><li> HBSS, standard, cioè con Ca 2 + e Mg 2 + (HBSS (w) </li></ol></li><li> A seconda del epitopo dell&#39;antigene di interesse per le applicazioni successive, utilizzare la papaina a base di tessuto neurale dissociazione Kit (P) o la tripsina a base di tessuto neurale dissociazione Kit (T). </li><li> Preparare le seguenti soluzioni dal tessuto neurale dissociazione Kit: <ol><li> Soluzione 2: aggiungere beta-mercaptoetanolo a 0,067 mM </li><li> Soluzione 4: sciogliere polvere in 0,7 ml di buffer di archiviazione (fornito nel kit), mescolare delicatamente (non vortex) </li><li> Enzyme Mix 1: Pipettare 1,9 mL Soluzione 2 e 50 ul di soluzione 1 (entrambi da kit) in un tubo C e incubare per 10-15 minuti a 37 ° C. Questo è sufficiente a dissociarsi 400 mg di tessuto cerebrale. </li></ol></li></ol> 2. Dissociare il tessuto nervoso <ol><li> Pesare tessuto cerebrale in una provetta contenente 1 ml HBSS (w / o) </li><li> Trasferimento cervello di topo nel tubo C contenente 1950 ml di Enzyme Mix preriscaldato 1. (NOTA: questo protocollo è scritto per ≤ 400 mg, ma volumi di soluzione può essere scalata a ospitare fino a 1600 mg di tessuto cerebrale per tubo C) </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_01&quot;. </li><li> Incubare con rotazione (4 giri al minuto usando un tubo Rotator MACSmix) per 15 minuti a 37 ° C. </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_02&quot;. </li><li> Durante la fase di dissociazione preparare 30 ml di Enzyme Mix 2 per 400 mg di tessuto con delicatezza mescolando 20 ml di soluzione 3 e 10 ml di soluzione 4. </li><li> Aggiungi Enzyme Mix 2 al tubo C attraverso il setto-sigillato tappo e mescolare delicatamente senza vortex. </li><li> Incubare la provetta C con rotazione per 10 minuti a 37 ° C in un incubatore. </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_03&quot;. </li><li> Incubare la provetta C con rotazione per 10 minuti a 37 ° C in un incubatore. </li><li> Centrifugare brevemente per raccogliere il campione al fondo della provetta. </li></ol> 3. Filtrazione <ol><li> Selezionare un filtro abbastanza grande per consentire il passaggio alle cellule di interesse. Per esempio, cellule di Purkinje sono troppo grandi per passare attraverso un filtro di 30 micron, ma Prominin-1 + cellule. </li><li> Utilizzare una pipetta suitable1000 microlitri per rimuovere le cellule formano il tubo C il setto-tappo sigillato e applicarlo al pre-filtro di separazione su un tubo di raccolta da 15 ml. (NOTA: per le dimensioni del campione più ampio utilizzare un tubo di raccolta da 50 ml). </li><li> Lavare il filtro con 10 ml di HBSS (w). </li><li> Centrifugare le cellule filtrata a 300 xg per 10 minuti a temperatura ambiente. </li><li> Risospendere il supernatent nel medio o tampone di scelta per le applicazioni successive. </li><li> Per rimuovere i detriti della mielina, utilizzare Beads rimozione mielina. </li></ol> 4. Risultato rappresentante: Si prega di vedere figure 1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="fig 1" /> Figura. 1 La dissociazione cervello con il dissociatore gentleMACS portato a 97% cellule vitali, come dimostra la citometria a flusso. <img src="/files/ftp_upload/1267/1267fig2.png" alt="fig 2" /> Figura. 2 foto microscopio Luce di formazione neurosfere dopo l&#39;ordinamento delle cellule magnetico utilizzando Anti-Prominin-1 microsfere dopo 7 giorni di coltivazione in MACS ® NeuroMedium integrato con MACS supplemento B27 PLUS. Le cellule sono state preparate dal CD1 cervello di topo (P3) con il tessuto neurale dissociazione Kit (P). <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="Fig. 3.5" /> Figura. 3 detriti mielina in cella singola sospensioni un notevole pericolo per l&#39;isolamento delle cellule, e la rimozione di detriti mielina da Perline rimozione mielina separazioni aumenta l&#39;efficienza delle cellule. Per le separazioni MACS con Anti-Prominin-1 microsfere, P22 cervello di topo era dissociato con il tessuto neurale dissociazione Kit (P). Cellule dalla cella singola sospensione sono state direttamente utilizzate per la separazione, o sono stati sottoposti al depauperamento della mielina con perline di rimozione mielina. Confrontando la separazione da campioni, senza e con la rimozione della mielina, dimostra che la purezza è maggiore per i campioni con la rimozione della mielina precedente.

<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>

Gli autori sono dipendenti di Miltenyi Biotec GmbH, Germania.

Il dissociatore gentleMACS facilita la preparazione standardizzata di singole cellule sospensioni dai tessuti neurali in un sistema chiuso. Neurali del tessuto kit dissociazione sono ottimizzati per preservare epitopi antigene necessaria per ulteriori applicazioni, come immunostainings e la separazione delle cellule immunomagnetica 1-5. In questo protocollo si mostra la dissociazione dolce enzimatica di cervello di topo utilizzando il dissociatore gentleMACS e il tessuto neurale dissociazione Kit (P), ottenendo nel 97% cellule vitali. 100 mg di rendimenti tra il tessuto neurale 5x10 1x10 6 e 7 celle, a seconda dell&#39;età del tessuto ospite. Il mirati Prominin-1 + sono state isolate le cellule utilizzando Anti-Prominin-1 microsfere e successivamente presi in cultura. Il protocollo è dimostrato di essere ancora più efficace con l&#39;uso aggiuntivo di Perline rimozione mielina quando si lavora con i tessuti neurali derivate da topi P7&gt;.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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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_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

All&#39;interno del sistema nervoso, centinaia di tipi di cellule neuronali e gliali sono stati descritti. Ogni tipo di cellule specifiche del cervello o del midollo spinale ha un repertorio di molecole di superficie cellulare, o determinanti molecolari, attraverso i quali può essere identificato e caratterizzato. Attualmente l&#39;identificazione delle cellule robuste e richiedono tecnologie di separazione a cella singola preparati da generare al tempo stesso limitare la morte cellulare e la distruzione delle proteine ​​di superficie caratteristico. Il dissociatore gentleMACS, se usato in combinazione con tripsina o la papaina kit a base di dissociazione, può efficacemente e delicatamente dissociare tessuto cerebrale, preservando epitopi dell&#39;antigene e limitare la perdita di cellule. Preparazione standardizzata di singola cellula sospensioni è ottenuta utilizzando tubi C e ottimizzato, programmi preimpostati gentleMACS. Una volta generato, cella singola sospensioni possono essere trattati con coniugati monoclonali come Anti-Prominin-1 microsfere, che identificano progenitori neurali, o ulteriormente purificato utilizzando perline di rimozione mielina.

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

Dissociare cellule di tipi di tessuto specifico richiede parametri specifici per aggitation tessuto 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 nerual è spiegato.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.4K Views.  Miltenyi Biotec,GmbH.  中文 日本語 

Video: 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.

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

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 ...

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.

L&#39;ottenimento di RNA di alta qualità da popolazioni di cellule singole nei tessuti umani cervello postmortem

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.

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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.

Generazione di tubuloidi renali umani da tessuti e urine

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

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolamento delle cellule endoteliali dal lume delle arterie carotidi di topo per esperimenti multi-omici a singola cellula

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

Trascrizione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore gentleMACS

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

L'ottenimento di RNA di alta qualità da popolazioni di cellule singole nei tessuti umani cervello postmortem

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

Generazione di topi derivati da cellule staminali pluripotenti indotte

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Generazione di tubuloidi renali umani da tessuti e urine

Isolamento delle cellule endoteliali dal lume delle arterie carotidi di topo per esperimenti multi-omici a singola cellula

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

Trascrizione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore gentleMACS

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

L'ottenimento di RNA di alta qualità da popolazioni di cellule singole nei tessuti umani cervello postmortem

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Generazione di tubuloidi renali umani da tessuti e urine

Isolamento delle cellule endoteliali dal lume delle arterie carotidi di topo per esperimenti multi-omici a singola cellula

Generazione di topi derivati da cellule staminali pluripotenti indotte