Pour travailler dans des conditions stériles, il est recommandé d&#39;effectuer toutes les étapes d&#39;une hotte à flux laminaire. 1. Matériaux <ul><li> Neural Tissue Kit de dissociation (P) (Composants: Solution 1,2, 3 et 4, de stockage tampon) </li><li> Pré-Séparation Filtres </li><li> (Facultatif) Perles de suppression myéline </li><li> Les bêta-mercaptoéthanol </li><li> gentleMACS dissociateur </li><li> C Tubes </li><li> Rotator Tube MACSmix </li><li> HBSS (w / o) </li><li> HBSS (w) </li></ul><ol><li> Préparer les solutions suivantes avant de commencer le protocole: <ol><li> Hanks solution saline tamponnée sans cations divalents Ca 2 + ou Mg 2 + (HBSS (w / o) </li><li> HBSS, standard, c&#39;est à dire avec Ca 2 + et Mg 2 + (HBSS (w) </li></ol></li><li> Selon l&#39;épitope d&#39;antigène d&#39;intérêt dans les applications ultérieures, utilisez soit la papaïne à base de tissus neuronaux dissociation Kit (P) ou de la trypsine à base de tissus neuronaux dissociation Kit (T). </li><li> Préparer les solutions suivantes de la dissociation des tissus neuronaux du kit: <ol><li> Solution 2: ajouter des bêta-mercaptoéthanol à 0,067 mM </li><li> Solution 4: dissoudre la poudre dans 0,7 ml de tampon de stockage (fourni dans le kit), mélanger délicatement (ne pas vortexer) </li><li> Mélanger une enzyme: Pipeter 1,9 ml de solution 2 et 50 ul de solution 1 (tous deux de kit) dans un tube C et incuber pendant 10-15 min à 37 ° C. Cela est suffisant pour dissocier de 400 mg de tissu cérébral. </li></ol></li></ol> 2. Dissociant le tissu neural <ol><li> Peser le tissu cérébral dans un tube contenant 1 ml de HBSS (w / o) </li><li> Transfert cerveau de souris dans le tube C contenant 1950 ul de mélange enzymatique préchauffé 1. (REMARQUE: ce protocole est rédigé pour ≤ 400 mg, mais les volumes de solution peut être adaptée pour accueillir jusqu&#39;à 1600 mg de tissu cérébral par Tube C) </li><li> Placer le tube C sur le dissociateur gentleMACS et exécutez le programme &quot;m_brain_01&quot;. </li><li> Incuber avec rotation (4 min en utilisant un tube Rotator MACSmix) pendant 15 min à 37 ° C. </li><li> Placer le tube C sur le dissociateur gentleMACS et exécutez le programme &quot;m_brain_02&quot;. </li><li> Pendant l&#39;étape de dissociation de préparer 30 pi de 2 Mix enzyme par 400 mg de tissu par un léger mélange de 20 ul de la solution 3 et 10 ul de la solution 4. </li><li> Ajouter 2 mélange enzymatique du métro C via le septum scellé bouchon et mélanger doucement sans vortex. </li><li> Incuber le tube C avec rotation pendant 10 min à 37 ° C dans un incubateur. </li><li> Placer le tube C sur le dissociateur gentleMACS et exécutez le programme &quot;m_brain_03&quot;. </li><li> Incuber le tube C avec rotation pendant 10 min à 37 ° C dans un incubateur. </li><li> Centrifuger brièvement pour prélever l&#39;échantillon au fond du tube. </li></ol> 3. Filtration <ol><li> Sélectionner un assez grand filtre pour permettre le passage vers les cellules d&#39;intérêt. Par exemple, les cellules de Purkinje sont trop grands pour passer à travers un filtre de 30 um, mais Prominin-1 + cellules. </li><li> Utiliser une pipette suitable1000 ul pour éliminer les cellules forment le tube C à travers le septum scellé bouchon et l&#39;appliquer à la pré-filtre de séparation sur un tube de prélèvement de 15 ml. (REMARQUE: pour des échantillons plus importants d&#39;utiliser un tube de prélèvement de 50 ml). </li><li> Laver le filtre avec 10 ml de HBSS (w). </li><li> Centrifuger les cellules filtrées à 300 xg pendant 10 min à température ambiante. </li><li> Reprendre le surnageant dans votre moyenne ou tampon de choix pour les applications ultérieures. </li><li> Pour enlever les débris de myéline, l&#39;utilisation Perles de suppression de myéline. </li></ol> 4. Résultat Représentant: S&#39;il vous plaît voir Figures 1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="La figure 1" /> Figure. 1 La dissociation du cerveau avec la dissociateur gentleMACS abouti à 97% de cellules viables, comme le montre l&#39;analyse des flux de cytométrie. <img src="/files/ftp_upload/1267/1267fig2.png" alt="La figure 2" /> Figure. 2 photo Microscope optique de la formation Neurosphère après tri cellulaire magnétique en utilisant Anti-Prominin-1 microbilles après 7 jours de culture dans MACS ® NeuroMedium complétée par MACS supplément B27 PLUS. Les cellules ont été préparées à partir de cerveau de souris CD1 (P3) en utilisant le tissu neural dissociation Kit (P). <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="figure 3.5" /> Figure. 3 débris de myéline dans une seule cellule suspensions altère considérablement l&#39;isolement cellulaire, et l&#39;enlèvement des débris de myéline par les perles de suppression myéline accroît les séparations de cellules d&#39;efficacité. Pour les séparations MACS utilisant Anti-Prominin-1 microbilles, P22 cerveau de souris a été dissociée en utilisant les tissus neuronaux dissociation Kit (P). Les cellules de la suspension à cellule unique ont été soit directement utilisé pour la séparation, ou ont été soumis à la myéline déplétion en utilisant des perles de suppression de myéline. En comparant la séparation à partir d&#39;échantillons avec et sans suppression de la myéline, démontre que la pureté est supérieure pour les échantillons avec l&#39;enlèvement de myéline précédente.

<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>

Les auteurs sont des employés de Miltenyi Biotec GmbH, Allemagne.

Le dissociateur gentleMACS facilite la préparation standardisée des suspensions unicellulaires à partir des tissus neuraux dans un système fermé. Neural Tissue Kits de dissociation sont optimisés pour préserver épitopes antigène nécessaire pour d&#39;autres applications comme les immunomarquages ​​et la séparation des cellules immuno 1-5. Dans ce protocole, nous montrons la dissociation enzymatique doux de cerveau de souris en utilisant les dissociateur gentleMACS et le tissu neural dissociation Kit (P), ce qui donne dans 97% des cellules viables. 100 mg de tissu neural entre les rendements 5x10 et 1x10 6 7 cellules, en fonction de l&#39;âge du tissu hôte. La cible Prominin-1 + cellules ont été isolées à l&#39;aide anti-Prominin-1 microbilles et ensuite repris dans la culture. Le protocole est indiqué pour être encore plus efficace avec l&#39;utilisation additionnelle de perles de suppression myéline quand on travaille avec les tissus nerveux provenant de souris&gt; P7.

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		<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

Dans le système nerveux, des centaines de types de cellules neuronales et gliales ont été décrits. Chaque type de cellule spécifique dans le cerveau ou la moelle épinière a un répertoire de molécules de surface cellulaire, ou déterminants moléculaires, grâce auquel il peut être identifiée et caractérisée. Actuellement, l&#39;identification de cellules robustes et les technologies de séparation nécessitent une seule cellule préparations destinées à être produites tout en limitant la mort cellulaire et la destruction des protéines de surface caractéristique. Le dissociateur gentleMACS, lorsqu&#39;il est utilisé en combinaison avec la trypsine ou la papaïne basé kits de dissociation, peut efficacement et en douceur dissocient le tissu cérébral, tout en préservant épitopes d&#39;antigènes et de limiter la perte de cellules. Préparation standardisée des suspensions unicellulaires est réalisé en utilisant les tubes C et optimisées, prédéfinies Programmes gentleMACS. Une fois produite, une seule cellule suspensions peuvent être traitées avec des conjugués monoclonaux comme anti-Prominin-1 microbilles, qui identifient les progéniteurs neuronaux, ou encore purifiée en utilisant des perles de suppression de myéline.

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

Dissocier les cellules de différents types de tissus spécifiques requiert des paramètres spécifiques pour aggitation tissus pour obtenir un volume élevé de viabilité, de cellules cultivables. Le Miltenyi gentleMACS dissociateur optimise cette tâche avec un simple, protocole pratique. Dans cette publication, l&#39;utilisation de cet appareil sur le tissu nerual est expliqué.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

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

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JoVE Journal

Biology

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Vidéo: 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.

Préparation des suspensions unicellulaires à partir de rate de souris avec le dissociateur gentleMACS

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Recherche

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.

Préparation standardisée des suspensions unicellulaires provenant de tissus pulmonaires de la souris en utilisant les dissociateur 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.

Obtention d&#39;ARN de haute qualité provenant des populations cellule unique de tissus post-mortem du cerveau de l&#39;homme

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.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Génération de tubuloïdes rénaux humains à partir de tissus et d’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 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).

Isolement de noyaux de tissu hépatique surgelé pour la multiomique unicellulaire

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.

Préparation de suspension unicellulaire de l’intestin de tilapia du Nil pour séquençage unicellulaire

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.

Isolement et analyse transcriptome de types de cellules végétales

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

Digestion cellulaire en deux étapes de l’artère carotide de souris

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.

Pleins feux sur l’auteur : Dissociation du tissu vasculaire et exploration de sous-groupes unicellulaires pour une thérapie ciblée 

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

Préparation d’une suspension unicellulaire à partir d’artères carotides de souris pour le séquençage unicellulaire

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 ...