Les auteurs tiennent à remercier le Dr Philip H. Schwartz, de la National des Ressources Humaines des cellules souches neurales à l&#39;Hôpital des Enfants, le comté d&#39;Orange Institut de recherche pour fournir des cultures hNSPC et le Dr Gary Bassell à l&#39;Université Emory d&#39;instruction initiale dans la technique de coloration parafilm.

Jour 1: Préparation des lamelles en verre Cellules allemande et l&#39;ensemencement Remarque: des lamelles de verre allemands sont souvent préférable pour la culture primaire des cellules souches neurales et les neurones. Nous utilisons le protocole de nettoyage suivantes pour améliorer l&#39;adhérence des cellules et se propager. Position des lamelles dans un rack petits qui permettra l&#39;accès de liquide aux deux côtés de la lamelle. Tout d&#39;abord, des lamelles incuber dans Liquinox 1% pendant 10 minutes, suivie de 3 lavages avec de l&#39;eau déminéralisée. Assurez-vous qu&#39;aucun des bulles de savon restent. Ensuite, glisse incuber dans HCl 1M pendant 30 minutes, suivie de 3 lavages avec de l&#39;eau déminéralisée. Lamelles à sec à 65 ° C pendant la nuit. Transfert des lamelles dans un plat en verre et autoclave pour stériliser. <ol><li> Dans une hotte de culture de tissus, placer des lamelles stériles dans une plaque de taille appropriée ainsi. </li><li> Manteau avec des lamelles de la laminine de l&#39;adhésion cellulaire. Lamelles Incuber dans 10 ug / ml de poly D-Lysine (PDL) dans l&#39;eau stérile pendant 5 minutes à température ambiante. Retirer lamelles PDL et incuber à 20 mg / ml dans la laminine EMEM pendant 4 heures ou toute la nuit, dans un 37 ° C de culture de tissu incubateur. <ol><li> Note: Se référer à l&#39;article de repiquage neuronaux humains Cellules souches ( <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ) et le comptage de l&#39;homme souches neurales l&#39;article Cells ( <a href="/index/Details.stp?ID=262">https://www.jove.com / index / Details.stp? ID = 262</a> ) pour apprendre à resuspendre et compter hNSPCs. </li></ol></li><li> Rincer la laminine revêtement des lamelles avec du PBS et les cellules de graines dans chaque lamelle contenant bien à une certaine densité (on utilise souvent une densité de placage initiale de 100,000-300,000 cellules / ml pour les cellules que nous sommes de placage pour immunomarquage). </li><li> Placez la plaque à 37 ° C de culture de tissu incubateur et permettent aux cellules d&#39;adhérer à lamelles (habituellement un minimum de 4 heures est requis). </li></ol> Jour 2: Fixation, perméabilisant, le blocage, et ajout d&#39;anticorps primaire à cellules Note: Nous utilisons le fixateur paraformaldéhyde 4% suivants afin de préserver la morphologie cellulaire. La recette comporte une transition de pH pour permettre à la paraformaldéhyde à passer en solution. Nous préférons cette méthode plutôt que l&#39;utilisation de la chaleur pour dissoudre paraformaldéhyde parce températures plus élevées peuvent conduire à la génération de l&#39;acide formique, ce qui peut augmenter la coloration de fond lors de l&#39;utilisation de fluorescence. Certaines composantes de l&#39;aide fixatif pour préserver la structure du cytosquelette (MgCl 2 et EGTA), tandis que d&#39;autres aident à maintenir la morphologie globale (saccharose). Fixateur paraformaldéhyde à 4% pour les cellules <table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Réactifs et étapes: </td><td> Montant pour 100 ml de fixateur: </td></tr><tr><td> L&#39;eau MilliQ </td><td> 75 ml </td></tr><tr><td> Paraformaldéhyde (peser dans la hotte) </td><td> 4 g </td></tr><tr><td> NaOH 10N, remuer et ajouter goutte à goutte jusqu&#39;à ce qu&#39;une solution efface </td><td> au besoin </td></tr><tr><td> PBS 10X </td><td> 10 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0,5 ml </td></tr><tr><td> 0,5 M EGTA </td><td> 2 ml </td></tr><tr><td> Saccharose </td><td> 4 g </td></tr><tr><td> HCl 6N, titrer à un pH de 7,4 </td><td> au besoin </td></tr><tr><td> L&#39;eau MilliQ </td><td> porter à 100 ml </td></tr><tr><td colspan="2"> Conserver à 4 ° C pendant 1 semaine. Chaude à 37 ° C avant utilisation </td></tr></tbody></table><ol><li> Sur le banc, retirez le support et ajouter fixateur préchauffée. Incuber pendant 5-15 minutes (en fonction de l&#39;antigène à détecter, on utilise habituellement 10 minutes). Effectuez cette et les étapes suivantes à la température ambiante. </li><li> Jeter le fixateur dans un récipient approprié, comme de nombreuses institutions gérer paraformaldéhyde comme déchets dangereux. Laver les cellules trois fois avec du PBS, à 5 minutes à chaque fois. Lors de la suppression de solutions à partir des cellules, faites attention que l&#39;aspiration ne se dessèche pas les cellules. Aussi, ont toujours la solution suivante sur la main d&#39;ajouter aux cellules avant de retirer la solution que les manteaux eux pour les cellules ne sont pas laissés à sécher. </li><li> Si l&#39;antigène d&#39;intérêt est à l&#39;intérieur de la cellule, la membrane cellulaire doit être rendue perméable pour permettre l&#39;entrée de l&#39;anticorps. Pour perméabiliser les cellules, utilisez un nouveau 0,3% de Triton X-100 solution dans du PBS. Pipeter lentement parce que Triton est visqueux, et assurez-vous que le détergent est complètement dissous dans du PBS avant d&#39;ajouter aux cellules. Perméabiliser les cellules pendant 5 minutes, suivi par le lavage des cellules 3 fois avec du PBS, à 5 minutes à chaque fois. </li><li> Les cellules doivent être traitées avec un agent de blocage pour empêcher la liaison non spécifique de l&#39;anticorps. Nous utilisons l&#39;albumine sérique bovine (BSA) comme agent de blocage. La solution de blocage est faite en dissolvant BSA 5% dans du PBS (sur une bascule ou des rotateurs, car cela prend du temps à se dissoudre), puis filtrer le solution d&#39;un filtre à seringue de 0,45 um. Bloc cellules dans 5% BSA / PBS solution pendant 1 heure à température ambiante. </li></ol> Diluer l&#39;anticorps primaire (qui est, espérons-spécifiques de la protéine d&#39;intérêt) à une concentration pré-déterminé de 1% BSA / PBS (préparé par dilution de 5% de BSA / PBS). Placez diluée d&#39;anticorps primaire (30 pi par 25 lamelle mm, 20 ul par 18 mm lamelle, 15 pi par 12 lamelle mm) sur une plaque de verre recouverts de parafilm. Ramassez la lamelle avec des cellules fixes, inverser telles que les cellules, face cachée, et mettez-le sur la solution d&#39;anticorps afin que les cellules sont en contact avec l&#39;anticorps. Placez une bande de parafilm seconde au cours de la construction entière. Incuber à 4 ° C pendant la nuit. Jour 3: Lavage, incubation anticorps secondaire, Stain nucléaire et de montage <ol><li> Retirez délicatement la bande de parafilm dessus et ramassez la lamelle, l&#39;inverser, et de le replacer dans la plaque avec du PBS dans chaque puits pour les lavages de sorte que les cellules sont vers le haut. </li><li> Laver les cellules trois fois avec du PBS, à 5 minutes à chaque fois. </li><li> Choisissez un anticorps secondaire qui permet de détecter l&#39;anticorps primaire, par exemple, si l&#39;anticorps primaire a été faite chez le lapin, l&#39;anticorps secondaire devraient reconnaître IgG de lapin. Prenez soin aussi de correspondre à l&#39;isotype d&#39;anticorps (IgG, IgM, etc.) Si vous utilisez plusieurs anticorps primaires, assurez-vous qu&#39;ils sont de différentes espèces ou isotypes, et le plan de détecter avec différents marqueurs attachés à l&#39;anticorps secondaire. Par exemple, vous pourriez utiliser un anti-lapin couplé à un fluorophore secondaire rouge avec un anti-souris couplé à un fluorophore secondaires vert. Incuber les cellules dans la solution d&#39;anticorps secondaire dilué à une concentration appropriée dans de la BSA 1% pendant 2 heures dans le noir à température ambiante, en utilisant la même technique que pour le primaire (étapes ne figurent pas dans la vidéo). Le volume d&#39;anticorps secondaire nécessaire pour l&#39;incubation peut être légèrement supérieure à celle de l&#39;anticorps primaire si le secondaire est stocké comme une solution de glycérol (40 pi par 25 mm lamelle, 30 pi par 18 mm lamelle, 25 pi par 12 mm lamelle) . <ul><li> Remarque: Si vous utilisez des molécules fluorescentes pour visualiser l&#39;anticorps secondaire, l&#39;échantillon doit être protégé de la lumière une fois de l&#39;anticorps secondaire a été ajoutée. Incuber dans l&#39;obscurité, ou dans une plaque aluminium couvert. </li></ul></li><li> Retirez délicatement les lamelles de la parafilm comme décrit pour l&#39;anticorps primaire. Laver les cellules deux fois avec du PBS, à 5 minutes à chaque fois, suivi par une incubation de 1 minute à 2 ug / ml colorant nucléaire Hoechst dilué dans du PBS (si une contre-coloration nucléaire est préférée). Si la tache est vieille et Hoechst, le signal est trop faible, vous pouvez augmenter le temps d&#39;incubation et / ou la concentration. Après incubation avec Hoechst, laver une fois avec du PBS pendant 5 minutes. </li><li> Les lamelles doivent être montées sur une lame avec les médias de montage pour une visualisation sur un microscope. Dans les cas où une molécule fluorescente est utilisé pour la visualisation de l&#39;anticorps secondaire, les médias de montage doit contenir des agents pour minimiser photoblanchiment. Nous utilisons Vectashield comme un média de montage pour la fluorescence. Placez une goutte de taille appropriée de Vectashield sur une lame de microscope (12 pi par 25 lamelle mm, 6 pi par 18 mm lamelle, 3 pi par 12 lamelle mm). </li><li> Soigneusement ramasser la lamelle et rincer le dos avec de l&#39;eau déminéralisée pour éliminer les sels (à partir du PBS). Tamponnez l&#39;excès d&#39;eau sur une serviette en papier ou Kimwipe, et jeter sur la goutte de Vectashield avec des cellules vers le bas. Placer la lamelle sur un angle et permettent de descendre lentement pour éviter les bulles d&#39;air de piégeage. Étiquette de la diapositive avec la date et de toute information sur l&#39;échantillon. </li><li> Autoriser des diapositives avec des lamelles de sécher (dans le noir), puis d&#39;aspiration hors Vectashield excès de la lamelle sur le pourtour. Les bords lamelle peut maintenant être scellé avec du vernis à ongles, si désiré (particulièrement utile si des lamelles à être visionnés sur un microscope inversé). En général, nous stockons nos diapositives dans un congélateur à -20 ° C. </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

Ce protocole décrit une procédure d&#39;immunomarquage hNSPCs qui minimise la quantité d&#39;anticorps nécessaire et donne une coloration cellulaire fiable. La procédure décrite est la meilleure pour les antigènes intracellulaires, mais peut être modifiée pour tache de molécules de surface cellulaire ou à améliorer la coloration du cytosquelette.

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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>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

immunocytochemistry: human neural stem cells

Immunocytochimie est une méthode très puissante et assez simple pour déterminer la présence, la localisation subcellulaire, et l&#39;abondance relative d&#39;un antigène d&#39;intérêt, le plus souvent une protéine, dans les cellules cultivées. Ce protocole présente un outil facile à suivre la série de mesures qui permettront aux chercheurs de conserver les anticorps primaires et secondaires tout en obtenant de haute qualité, reproductibles et qualitative des données quantitatives de leur coloration. Il ya deux aspects de ce protocole qui aidera à conserver le volume d&#39;anticorps nécessaire pour la coloration. D&#39;une part, les cellules sont cultivées sur de petites lamelles circulaires qui sont placés dans les puits d&#39;une plaque de culture de tissus. Après fixation, les cellules sur des lamelles peuvent être retirés dans les puits de la plaque. Pour la coloration des anticorps, la lamelle avec des cellules est inversée sur une petite goutte de solution d&#39;anticorps sur parafilm et est recouvert d&#39;un second morceau de parafilm pour éviter le dessèchement. En utilisant cette méthode, seulement ~ 25 pl de solution d&#39;anticorps est nécessaire pour chaque lamelle (ou échantillons) doivent être colorées. Ce protocole décrit immunomarquage des souches neurales humaines / cellules précurseurs (hNSPCs), mais peut être utilisé pour de nombreux autres types cellulaires.

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

Immunocytochimie est une méthode puissante pour déterminer la présence, la localisation subcellulaire, et l&#39;abondance relative d&#39;un antigène d&#39;intérêt dans les cellules cultivées. Ce protocole présente un outil facile à suivre la série d&#39;étapes qui permettent de conserver les anticorps et tirer le meilleur parti de sa coloration.

Immunocytochimie: des cellules souches humaines

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

Research

JoVE Journal

Biology

27.3K vues.  University of California, Irvine (UCI). Immunocytochimie est une méthode puissante pour déterminer la présence, la localisation subcellulaire, et l'abondance relative d'un antigène d'intérêt dans les cellules cultivées. Ce protocole présente un outil facile à suivre la série d'étapes qui permettent de conserver les anticorps et tirer le meilleur parti de sa coloration.

Vidéo: Immunocytochemistry: Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagation des souches embryonnaires humaines (ES) Cellules

Recherche

Biologie

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfection des cellules souches humaines à l&#39;Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Comptage des cellules souches humaines

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Le repiquage des cellules souches humaines

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Souris épidermique Neural Stem Cell Crest (EPI-NCSC) cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congélation et de décongélation cellules souches embryonnaires humaines

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfection et Nucleofecting homme cellules souches pluripotentes induites

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...