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Prima dell&#39;isolamento dell&#39;ippocampo Prima di iniziare l&#39;isolamento dell&#39;ippocampo, assicurarsi che tutti gli strumenti sono sterili. Spray giù il cappuccio della cultura con il 70% di etanolo, e atto gli strumenti all&#39;interno della cappa. Avrete bisogno di 6 - e 10 cm di piastre di Petri, sterile poli-L coprioggetto vetro rivestito, pipette e le punte, pipette monouso, e un pipettatore elettrica. Da questo punto in poi, ricordatevi di usare correttamente la tecnica sterile. <ol><li> Accendere il bagno d&#39;acqua e fare in modo che viene riscaldato fino a 37 ° C. </li><li> Le seguenti soluzioni che vengono conservati a 4 ° C saranno necessarie: <ul><li> Media modificato Eagle (MEM) </li><li> Neurobasal </li><li> Soluzione salina tamponata di Hank (HBSS) </li><li> Soluzione tampone borato </li><li> Piruvato di sodio soluzione </li><li> Sterile filtrata soluzione di glucosio al 20% in acqua distillata </li><li> Assicurati di spruzzare tutte le bottiglie con il 70% di etanolo prima di metterli nel cofano. </li></ul></li><li> Prendete queste soluzioni congelato dal freezer e metterli in un bagno di acqua riscaldata: <ul><li> Un&#39;aliquota di 5 ml di siero di cavallo </li><li> A 1 ml di aliquota B-27 supplemento </li><li> A 1 ml di aliquota 100X antibiotico (penicillina più streptomicina) </li><li> E di 0,5 ml aliquota di L-glutammina </li></ul></li><li> Dopo le soluzioni sono scongelati, li tolga dal bagno d&#39;acqua, spruzzi giù con il 70% di etanolo, e metterli nel cofano. Ora il medium placcatura e Neurobasal possono essere preparati. </li><li> Dopo le soluzioni sono preparate, tappo i contenitori ermeticamente e metterli nella vasca 37 ° C per riscaldarsi durante l&#39;esecuzione l&#39;isolamento dell&#39;ippocampo. </li><li> Inoltre, prendere una aliquota della proteasi tripsina (2,5%) dal freezer e metterlo in bagno d&#39;acqua. Tripsina sarà digerire l&#39;ippocampo sezionato, che sarà isolato nella fase successiva. </li><li> Prendete un tubo 15ml conica, riempirlo con HBSS, e l&#39;etichetta con il gruppo di trattamento. E &#39;qui che isolato dell&#39;ippocampo saranno raccolti nella fase successiva. </li></ol> Isolamento dell&#39;ippocampo <ol><li> Per iniziare l&#39;isolamento dell&#39;ippocampo, assicurarsi che i cuccioli appena nati sono pulite e hanno avuto la loro band il latte, placente e cordoni ombelicali rimosso dalla madre. </li><li> Posizionare i cuccioli in piastre di Petri, spruzzare con il 70% di etanolo per la pulizia, e metterli nel cofano. Gli animali sono l&#39;eutanasia immediatamente prima coltura. </li><li> Per la sterilizzazione ulteriore prendere uno cucciolo dal piatto, immergere il cucciolo in etanolo al 70%, e poi in due lavaggi di HBSS sterile. </li><li> Rimuovere la testa dal corpo con le forbici. Utilizzando le forbici stesso, tagliare attraverso la pelle e il cranio. </li><li> Utilizzando un paio di pinzette bene, la buccia del cranio di distanza dal cervello e mettere il cervello in una piccola scatola di Petri che contiene una piccola quantità di HBSS sterile. </li><li> Staccare gli emisferi cerebrali. L&#39;ippocampo è una piccola struttura a forma di cavalluccio marino nel lobo temporale mediale. </li><li> Rimuovere l&#39;ippocampo e il luogo in 3 ml di HBSS in una provetta da 15 ml. Ripetere questi passaggi con ogni cucciolo, e svolge ogni ippocampo isolato nel tubo da 15 ml. Ora, è tempo di dissociare il tessuto di singole cellule. </li></ol> Dissociazione delle cellule dell&#39;ippocampo <ol><li> Dopo tutto dell&#39;ippocampo sono state isolate, riempire il tubo 15 ml a 4,5 ml con HBSS. </li><li> Rimuovere la tripsina dal bagno d&#39;acqua, spruzzare con l&#39;etanolo, e posto nel cofano. Aggiungere 0,5 ml di tripsina al tubo, e incubare per 15 minuti a 37 ° C. </li><li> Nel cofano, rimuovere il HBSS / tripsina soluzione dal tubo con una pipetta sterile, facendo attenzione a non disturbare il dell&#39;ippocampo che si sono insediati sul fondo della provetta. Aggiungere 5 ml di HBSS al tubo e miscelare delicatamente. Incubare a 37 ° C per 5 minuti. </li><li> Ripetere questo passaggio due volte, la rimozione del vecchio HBSS e la sostituzione con una soluzione fresca. </li><li> Prelevare un &#39;aliquota di DNAsi I fuori dal freezer. Aggiungere 0,5 ml di DNasi al dell&#39;ippocampo HBSS in 4,5 ml. La DNAsi è aggiunto a promuovere l&#39;inattivazione dell&#39;enzima. </li><li> Triturare la soluzione (o pipetta su e giù) finché non è omogeneo. Fare attenzione a non introdurre bolle in omogenato. </li><li> Determinare la vitalità cellulare con il metodo trypan esclusione blu. </li><li> Prendete un piatto di 10 centimetri Petri contenente 6-8 vetrini, e aggiungere 10 ml di terreno placcatura. Pipettare il numero desiderato di cellule al piatto contenente il coprioggetto. Agitare delicatamente per disperdere le cellule, e assicurarsi che il coprioggetto non si sovrappongano. </li><li> Permettono alle cellule di attaccare per 2-4 ore in un umidificata a 37 ° C incubatore con 5% di CO 2. Dopo aver confermato che le cellule sono vitali e hanno attaccato, trasferire i coprioggetti ai piatti singoli contenenti terreno Neurobasal. Luogo questi piatti nuovamente dentro l&#39;incubatrice. </li><li> Una volta alla settimana, sostituire un terzo del mezzo con freschi di media Neurobasal e le cure sperimentali, se necessario. Ora, le proprietà funzionali di queste cellule in coltura sono pronti per essere studiati. </li></ol> Fura-2 Imaging di calcio di neuroni dell&#39;ippocampo <ol><li> Dopo colture di neuroni dell&#39;ippocampo sono state in vitro per 48 ore (ma non prima), gli esperimenti di imaging di calcio può avere inizio. A questo punto, questi neuroni dovrebbero avere iniziato a estendere i processi. La fascia di età ottimale per l&#39;imaging calcio è dal primo giorno in vitro 3 a 7. </li><li> Culture di carico con Fura-2 AM per 30 minuti a 37 ° C, dopo di che può essere ripreso sotto un obiettivo 20X usando una lampada ad arco allo xeno per eccitare il colorante indicatore di calcio e una fotocamera da 512 bit ccd, che campioni di ogni 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>

Questo protocollo è stato sviluppato come una modifica del lavoro seminale di Gary Banker e Kim Goslin 1. Questo libro è una risorsa essenziale per chiunque sia interessato a cellule di coltura - non solo il neurone arricchito di culture descritte nel protocollo corrente. I tre fattori più critici nella coltura delle cellule sono: la sterilità, la velocità, la scelta del mezzo utilizzato. Sterilità - un flusso laminare / cappa sterile, condizioni asettiche, e una sterile incubatore sono obbl...

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<td>Antibiotic antimitotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240062</td>
<td>&nbsp;</td>
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<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504044</td>
<td>Serum free</td>
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<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>
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<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
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<td>HBSS (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14185052</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630080</td>
<td>&nbsp;</td>
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<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>&nbsp;</td>
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<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>
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primary culture of hippocampal neurons from p0 newborn rats

Le proprietà fisiologiche dei neuroni dell&#39;ippocampo sono comunemente indagati, soprattutto per il coinvolgimento dell&#39;ippocampo nell&#39;apprendimento e nella memoria. Primaria coltura delle cellule dell&#39;ippocampo neuroscienziati permette di esaminare le attività e le proprietà dei neuroni in singole cellule e del livello singola sinapsi. In questo video ci dimostra come isolare e coltivare cellule primarie di ippocampo di ratti neonati. L&#39;ippocampo possono essere isolati gli uni dagli animali appena nati nel più breve 2-3 minuti, e le culture può essere mantenuta per un massimo di due settimane. Ci sarà anche brevemente illustrare come utilizzare questi neuroni dell&#39;ippocampo per l&#39;imaging di calcio raziometrico. Anche se questo protocollo descrive il processo per l&#39;ippocampo, con poca o nessuna modifica, può essere applicato ad altre regioni del cervello.

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 dissezione e la crescita delle cellule da una zona del cervello individuale facilita l&#39;investigazione dei parametri cellulari e fisiologici. Descriviamo un metodo per la coltura di cellule primarie che produce neurone arricchito di culture in un ambiente privo di siero.

Cultura primaria di neuroni dell&#39;ippocampo da P0 ratti neonati

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 Views.  Michigan State University (MSU). La dissezione e la crescita delle cellule da una zona del cervello individuale facilita l'investigazione dei parametri cellulari e fisiologici. Descriviamo un metodo per la coltura di cellule primarie che produce neurone arricchito di culture in un ambiente privo di siero.

Video: 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 trasfezione di neuroni dell&#39;ippocampo

Ricerca

Biologia

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.

Immagini dal vivo di Dense-core Vescicole nella Primaria neuroni coltivati ​​ippocampale

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.

Cultura primario di miociti adulti Cuore 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.

Dissezione di giro dentato ippocampale di topo adulto

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%.

Isolamento e la coltura primaria di cellule epatiche di ratto

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.

Isolamento e colture primarie di cellule di topo aortica endoteliali

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.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

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.

Isolamento e cultura delle cellule endoteliali aortiche primarie dai suini in miniatura

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.

Isolamento e differenziazione dei preadipociti bianchi e marroni primari dai topi appena nati

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.

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

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