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Avant l&#39;isolement hippocampique Avant le début de l&#39;isolement de l&#39;hippocampe, assurez-vous que tous les outils sont stériles. Vaporiser le bas de la hotte de la culture à l&#39;éthanol 70%, et place les outils à l&#39;intérieur du capot. Vous aurez besoin de 6 - et 10-cm boîtes de Pétri, stérile poly-L lamelles de verre enduit, pipettes et des conseils, des pipettes jetables, et une pipette électrique. A partir de ce moment, pensez à utiliser une technique stérile correcte. <ol><li> Mettez le bain d&#39;eau et assurez-vous qu&#39;il est chauffé à 37 ° C. </li><li> Les solutions suivantes qui sont conservés à 4 ° C seront nécessaires: <ul><li> Eagle modifié Medium (MEM) </li><li> Neurobasal </li><li> Hank solution saline tamponnée (HBSS) </li><li> Solution tampon borate </li><li> Solution de pyruvate de sodium </li><li> Filtration stérile solution de glucose à 20% dans l&#39;eau distillée </li><li> Assurez-vous de pulvériser toutes les bouteilles vers le bas avec 70% d&#39;éthanol avant de les placer dans la hotte. </li></ul></li><li> Prenez ces solutions congelées du congélateur et les placer dans un bain d&#39;eau chauffée: <ul><li> Une partie aliquote de 5 ml de sérum de cheval </li><li> Une partie aliquote de 1 ml de B-27 complètent </li><li> Une partie aliquote de 1 ml de 100X antibiotiques (pénicilline plus streptomycine) </li><li> Et une partie aliquote 0,5 ml de L-glutamine </li></ul></li><li> Après les solutions sont décongelés, les sortir du bain d&#39;eau, les arroser avec de l&#39;éthanol à 70%, et les placer dans la hotte. Maintenant, le support de placage et Neurobasal peuvent être préparés. </li><li> Après les solutions sont préparées, le bonnet d&#39;contenants hermétiquement et placez-les dans le bain à 37 ° C pour se réchauffer tout en effectuant l&#39;isolement hippocampe. </li><li> En outre, prélever une aliquote de la trypsine protéase (2,5%) hors du congélateur et le placer dans l&#39;eau du bain. La trypsine va digérer l&#39;hippocampe disséqué, qui sera isolé dans l&#39;étape suivante. </li><li> Prenez un tube de 15ml conique, le remplir avec du HBSS, et c&#39;est l&#39;étiquette avec le groupe de traitement. C&#39;est ici que hippocampes isolées seront recueillies à l&#39;étape suivante. </li></ol> Isolement hippocampe <ol><li> Pour commencer l&#39;isolement de l&#39;hippocampe, assurez-vous que les nouveau-nés sont propres et ont eu leurs bandes de lait, les placentas et cordons ombilicaux enlevés par leur mère. </li><li> Placez les chiots dans une boîte de Pétri, de pulvérisation avec de l&#39;éthanol à 70% pour nettoyer, et les placer dans la hotte. Les animaux sont euthanasiés immédiatement avant la culture. </li><li> Pour la stérilisation en outre, prendre un chiot de l&#39;antenne, tremper le chiot en éthanol de 70%, puis en deux lavages de HBSS stérile. </li><li> Retirez la tête du corps avec des ciseaux. En utilisant les ciseaux même, couper à travers la peau et le crâne. </li><li> En utilisant une paire de pinces fines, pelez le crâne loin du cerveau et la place du cerveau dans une petite boîte de Petri contenant une petite quantité de HBSS stérile. </li><li> Peler les hémisphères cérébraux. L&#39;hippocampe est une petite structure en forme d&#39;hippocampe-dans le lobe temporal médial. </li><li> Retirez l&#39;hippocampe et le lieu dans 3 ml de HBSS dans un tube de 15 ml. Répétez ces étapes avec chaque chiot, et placer chaque hippocampe isolés dans le tube de 15 ml. Maintenant, il est temps de dissocier le tissu à des cellules individuelles. </li></ol> Cellules hippocampiques de dissociation <ol><li> Après tout hippocampes ont été isolés, remplir le tube de 15 ml à 4,5 ml avec HBSS. </li><li> Retirez la trypsine du bain d&#39;eau, pulvérisation avec de l&#39;éthanol, et sa place dans le capot. Ajouter 0,5 ml de trypsine dans le tube et incuber pendant 15 minutes à 37 ° C. </li><li> Dans la hotte, enlever la solution HBSS / trypsine du tube avec une pipette stérile, en prenant soin de ne pas déranger les hippocampes qui se sont installés au fond du tube. Ajouter 5 ml de HBSS dans le tube et agiter doucement. Incuber à 37 ° C pendant 5 minutes. </li><li> Répétez cette étape deux fois, en supprimant l&#39;ancienne HBSS et les remplacer par une solution fraîche. </li><li> Prenez une aliquote de DNAse I du congélateur. Ajouter 0,5 ml de DNase à l&#39;hippocampe en 4,5 ml de HBSS. La DNase est ajoutée afin de promouvoir l&#39;inactivation des enzymes. </li><li> Triturer la solution (ou une pipette haut et bas) jusqu&#39;à ce qu&#39;elle soit homogène. Soyez prudent de ne pas introduire des bulles dans l&#39;homogénat. </li><li> Déterminer la viabilité des cellules avec la méthode exclusion du bleu trypan. </li><li> Prenez une boîte de Petri de 10 cm contenant 6 à 8 lamelles, et ajouter 10 ml de milieu de placage. Pipeter le nombre souhaité de cellules de la boîte contenant les lamelles. Agiter doucement pour disperser les cellules, et assurez-vous que les lamelles ne se chevauchent pas. </li><li> Laisser les cellules se fixer pendant 2-4 heures dans un incubateur humidifié 37 ° C avec 5% de CO 2. Après avoir vérifié que les cellules sont viables et ont fixé, le transfert des lamelles aux plats individuels contenant milieu Neurobasal. Placer ces plats de retour dans l&#39;incubateur. </li><li> Une fois par semaine, remplacez un tiers de la moyenne avec milieu Neurobasal frais et des traitements expérimentaux, au besoin. Maintenant, les propriétés fonctionnelles de ces cellules en culture sont prêts à être étudiés. </li></ol> Fura-2 d&#39;imagerie calcique des neurones de l&#39;hippocampe <ol><li> Après des cultures de neurones hippocampiques ont été in vitro pendant 48 heures (mais pas plus tôt), des expériences d&#39;imagerie calcique peut commencer. À ce stade, ces neurones doivent avoir commencé à étendre les processus. La tranche d&#39;âge optimal pour l&#39;imagerie du calcium est de jour in vitro de 3 à 7. </li><li> Cultures de charge avec Fura-2 heures 30 minutes à 37 ° C, après quoi ils peuvent être visualisés sous un objectif 20X en utilisant une lampe à arc Xenon pour exciter le colorant indicateur de calcium et d&#39;une caméra CCD de 512 bits, qui tous les échantillons 150ms. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells, 2nd Edition. , (1998).</li></ol>

Ce protocole a été développé comme une modification de l&#39;œuvre séminale de Gary Banquier et Kim Goslin 1. Ce livre est une ressource essentielle pour quiconque s&#39;intéresse à la culture de cellules - non seulement les cultures de neurones enrichi décrites dans le protocole actuel. Les trois facteurs les plus critiques dans la culture cellulaire sont: la stérilité, la vitesse, le choix du support utilisé. Stérilité - un écoulement laminaire / hotte stérile, des conditions d&#...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Antibiotic antimitotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240062</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504044</td>
<td>Serum free</td>
</tr>
<tr>
<td>Boric Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B6768</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25</td>
<td>&nbsp;</td>
<tr>
<td>Fura-2, AM cell permeant</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>F1221</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14185052</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630080</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>51200038</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12348017</td>
<td>Without phenol red</td>
</tr>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
<tr>
<td>Pyruvic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Tetraborate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 2.5% (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090046</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of hippocampal neurons from p0 newborn rats

Les propriétés physiologiques des neurones de l&#39;hippocampe sont couramment étudiés, notamment en raison de l&#39;implication de l&#39;hippocampe dans l&#39;apprentissage et la mémoire. Primaire culture cellulaire hippocampique permet neuroscientifiques d&#39;examiner l&#39;activité et les propriétés des neurones à la cellule individuelle et le niveau de la synapse unique. Dans cette vidéo, nous allons démontrer comment isoler et de cultiver des cellules primaires d&#39;hippocampe de rats nouveau-nés. L&#39;hippocampe peut être isolée de chaque animal nouveau-né dans le plus court de 2 à 3 minutes, et les cultures peuvent être maintenus pendant jusqu&#39;à deux semaines. Nous allons aussi brièvement démontrer comment l&#39;utilisation de ces neurones de l&#39;hippocampe pour l&#39;imagerie calcique ratiométrique. Alors que ce protocole décrit le processus de l&#39;hippocampe, avec peu ou aucune modification, il peut être appliqué à d&#39;autres régions du cerveau.

Watch this Scientific Journal Video about Primary Culture of Hippocampal Neurons from P0 Newborn Rats at JoVE.com

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

La dissection et la croissance de cellules provenant d&#39;une zone du cerveau individuel facilite enquête sur les paramètres cellulaires et physiologiques. Nous décrivons un procédé de culture de cellules primaires qui produit des cultures enrichies en neurones dans un environnement dépourvu de sérum.

Culture primaire de neurones de l&#39;hippocampe de rats nouveau-nés P0

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

primary-culture-of-hippocampal-neurons-from-p0-newborn-rats

Research

JoVE Journal

Biology

76.5K vues.  Michigan State University (MSU). La dissection et la croissance de cellules provenant d'une zone du cerveau individuel facilite enquête sur les paramètres cellulaires et physiologiques. Nous décrivons un procédé de culture de cellules primaires qui produit des cultures enrichies en neurones dans un environnement dépourvu de sérum.

Vidéo: Primary Culture of Hippocampal Neurons from P0 Newborn Rats

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

Gene-gun transfection de neurones de l&#39;hippocampe

Recherche

Biologie

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagerie en temps réel du noyau dense de vésicules cultures primaires neurones de l&#39;hippocampe

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Culture primaire des adultes myocytes cœur de rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissection de l&#39;hippocampe gyrus denté de souris adulte

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

L&#39;isolement et la culture primaire de cellules de foie de rat

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolement et culture des cellules endothéliales aortiques primaires des porcs miniatures

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Isolement et différenciation des préadipocytes primaires blancs et bruns de souris nouveau-nées

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolement et culture de kératinocytes oraux primaires du palais de souris adulte

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...