Parte I. Preparazione per la chirurgia <ol><li> Il giorno prima della chirurgia ratto, accertarsi che tutte le soluzioni sono fatti, ma non aggiungere alcun enzimi ancora. Buffer A, B e il tampone di lavaggio dovrebbero essere filtrate per la sterilità prima di stoccaggio. I tamponi dovrebbero essere conservato a 4 ° C. </li><li> 6-pozzetti colture di tessuti deve essere placcato con 10-20 mg / mL laminina in 2 ml 1XSFM il giorno prima come bene, e deve essere conservato a 37 ° C in un incubatore CO 2. </li><li> Il giorno della chirurgia, preparare gli strumenti chirurgici mediante sterilizzazione la pinza, forbici, pinze e con il 70% di etanolo e lasciarle asciugare su carta assorbente. </li><li> Una siringa da 1 ml riempita con 0,5 ml di soluzione di eparina (90 U / ml) è pronto. </li><li> Ora è buona norma lavare l&#39;apparato perfusione eseguendo attraverso 70% di etanolo, utilizzando la pompa peristaltica in senso inverso. Una volta finito il risciacquo con etanolo, sciacquare l&#39;apparato con il doppio di acqua distillata, ed infine risciacquare con tampone A. Il flusso attraverso viene raccolta in un bicchiere posto in una vasca d&#39;acqua a 37 ° C. </li><li> Per preparare l&#39;apparato perfusione puliti, riempire il tubo diritto siringa con 40 ml di tampone A e il tubo di sinistra siringa con 40 ml di tampone B. </li><li> Primo il tubo perfusione alla punta del catetere, consentendo tampone A a fluire attraverso l&#39;apparecchio. Ricarica al buffer una siringa al livello 40 ml. Assicurarsi che non vi siano bolle d&#39;aria intrappolate nel tubo dopo questo passaggio. </li><li> Ora, chiudere il buffer di una valvola di rubinetto siringa e ripetere il processo con tampone B tale che il tubo di perfusione è ora vaccinati con tampone B. </li><li> Infine, l&#39;apparato di perfusione è lasciato con la B Buffer rubinetto valvola aperta, ma con il flusso smesso di utilizzare il regolatore sulla linea IV. </li><li> Scartare il tampone di flusso, anche se nel bicchiere di raccolta. </li><li> L&#39;ultima cosa da fare prima della chirurgia è quello di aggiungere gli enzimi necessari per la soluzione stock, e quindi filtrare la soluzione enzimatica così come le altre soluzioni sono state filtrate il giorno prima. Una volta filtrata, questa soluzione può essere lasciato a temperatura ambiente. </li></ol> Parte II. The Rat dissezione cuore <ol><li> A seguito di protocollo standard eutanasia il topo con isoflurano in una camera a gas. </li><li> Sterilizzare l&#39;area incisione sul torace con il 70% di etanolo. </li><li> Torace aperto con le forbici e esporre il cuore. </li><li> Iniettare ventricolo sinistro con 0,5 mL di soluzione di eparina (90 U / mL). </li><li> Rimuovere il cuore con grande porzione di aorta intatto, posto a freddo Buffer ghiaccio B. </li></ol> Parte III. Miociti isolamento <ol><li> Un cuore di ratto tipico dovrebbe produrre una frazione alta a forma di bastoncello miociti (al contrario di cellule morte arrotondate) se la seguente procedura è correttamente rispettato. </li><li> Per iniziare, morsetto aorta al catetere. La punta del catetere non deve essere spinta troppo in là nel cuore di assicurare una buona perfusione del cuore attraverso l&#39;arteria coronaria. </li><li> Una volta bloccato, legare l&#39;aorta al catetere con una sutura. </li><li> Quindi, aprire il regolatore linea IV per consentire un ritmo veloce a goccia (~ 60 gocce / min) e permettere B tampone per lavare il cuore. </li><li> Durante il lavaggio tampone B, utilizzare le forbici piccole e pinze per rimuovere gli atri e qualsiasi tessuto adiposo o del polmone aggrappati al cuore. </li><li> Dopo tampone B è finito, passare il flusso di tampone A. </li><li> Mentre tampone A è permesso flusso per 5 minuti, alcuni piccoli compiti dovrebbero essere rapidamente eseguita. </li><li> In primo luogo, scaldare la soluzione enzimatica in un bagno d&#39;acqua a 37 ° C. </li><li> Infine, quando tampone A è esaurita dalla siringa (ma ancora presente nel tubo), carico di 50 ml di soluzione enzimatica in siringa Un dell&#39;apparato perfusione </li><li> Ora è necessario eliminare tutti i flow-through dal bicchiere raccolta nel bagno d&#39;acqua (pipettando), fino a quando la soluzione enzimatica cuore perfusione. </li><li> Non appena la soluzione enzimatica perfusione del cuore, attivare la pompa peristaltica che è impostato per trasferire la soluzione enzimatica dal bicchiere di raccolta per reintegrare siringa A. </li><li> Lasciare che la soluzione enzimatica a fluire attraverso il cuore per 10 min. Come il cuore digerisce, comincia a guardare gonfio. </li><li> Aggiungere 37,5 ml di 0,1 M CaCl 2 alla soluzione enzimatica in una siringa per dare un&#39;efficace concentrazione di 0,1 mM Ca 2 +. </li><li> Dopo 10 minuti aumentare la concentrazione di calcio a 0,2 mm con l&#39;aggiunta di un ulteriore 50 ml di 0,1 M CaCl 2 a siringa A e lasciare che la perfusione proseguire per altri 10 minuti. </li><li> Tagliare i ventricoli e trasferimento in un piccolo becher sterile contenente 20 ml di soluzione enzimatica. </li><li> Nel becher aumentare la concentrazione di calcio a 0,4 mm con l&#39;aggiunta di 40 microlitri di più di 0,1 M CaCl 2. </li><li> Tritate dolcemente il cuore in 10 o più pezzi con un paio di piccole forbici sterili. </li><li> Incubare il bicchiere per 5 minuti a 37 ° C con dolce dondolio. </li><li> Aggiungere 40 ml di 0,1 M CaCl 2 adare effettiva concentrazione di 0,6 mM Ca 2 + e delicatamente triturare i pezzi del cuore con una pipetta di plastica trasferimento 3-5 volte. </li><li> Dopo l&#39;incubazione per altri 5 minuti a 37 ° C, aggiungere 40 ml di 0,1 M CaCl 2 e delicatamente triturare come prima. </li><li> Separare miociti digerito singolo non digerito tessuto connettivo con filtrazione attraverso una sterile 500 mesh micron. </li><li> Permettono alle cellule di stabilirsi in un tubo da 50 ml per 10 minuti a temperatura ambiente. </li><li> Gettare il surnatante con una pipetta di trasferimento. </li><li> Risospendere delicatamente le cellule in soluzione di lavaggio # 1 e permettono alle cellule di accontentarsi di 10-20 minuti a temperatura ambiente. </li><li> Mentre le cellule si stanno stabilendo, prendere una piccola aliquota, e utilizzare questo tempo come occasione per valutare la qualità e la vitalità delle cellule in sospensione sotto un microscopio invertito. Una buona preparazione avrà una percentuale elevata (&gt; 80%) di asta come le cellule con striature croccante. </li><li> Una volta che le cellule si sono stabiliti, eliminare le cellule surnatante e risospendere delicatamente in soluzione di lavaggio # 2. Lasciate che le cellule stabilirsi a temperatura ambiente, che deve ancora prendere 10-20 minuti, e scartare il surnatante. </li><li> Ripetere il processo di lavaggio ancora una volta con tampone di lavaggio # 3, e le celle saranno pronti per la cultura. </li></ol> Parte IV. Coltura cellulare miociti <ol><li> Risospendere delicatamente le cellule in media il 5% del siero. Prima di trasferire le cellule a piastre di coltura di tessuto, lavare i piatti con 1x SFM. Trasferire le cellule ad una densità desiderata e incubare in un CO 2 di coltura tissutale incubatore per 3-5 ore. </li><li> Passa i media a 1x SFM. </li><li> Cellule possono essere coltivate per un massimo di quattro giorni e utilizzati come richiesto per gli esperimenti. </li></ol> Parte V. Rappresentante Risultati / Esito Ci dovrebbe essere oltre il 70% miociti cuore vivono sotto microscopio invertito quando il protocollo è fatto correttamente. <img src="/files/ftp_upload/1308/fig1.jpg" alt="figura 1" /> Tabella 1: Ricetta Soluzione per 1 L 10X Ca 2 + senza Buffer KH: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentrazione finale </td></tr><tr><td> NaCl </td><td> 68,96 g </td><td> 1180 mm </td></tr><tr><td> KCl </td><td> 3,58 g </td><td> 48 mm </td></tr><tr><td> HEPES </td><td> 59,58 g </td><td> 250 mm </td></tr><tr><td> K 2 HPO 4 </td><td> 2,85 g </td><td> 12,5 mm </td></tr><tr><td> MgSO 4 </td><td> 3,08 g </td><td> 12,5 mm </td></tr></tbody></table> Tabella 2: Ricetta Soluzione tampone per 1 L A: Aggiungere 1,98 g di glucosio a 100 ml di buffer 10X KH e portare a volume finale di 1 L. Regolare osmolarità a condizione fisiologia vicino (~ 300) con glucosio. Portare a pH 7,4 con NaOH. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> Massa </td><td> Concentrazione finale </td></tr><tr><td> KH Buffer 10X </td><td> 100 ml </td><td> 100 ml </td></tr><tr><td> Glucosio </td><td> 1,98 g </td><td> 11 mm </td></tr><tr><td> Portare a 1 L </td><td></td><td></td></tr><tr><td> Portare a pH 7,4 con NaOH </td><td></td><td></td></tr><tr><td colspan="3"> Regolare osmolarità quasi a condizione fisiologica (~ 300) con glucosio. </td></tr></tbody></table> Tabella 3: Ricetta Soluzione tampone enzimatico <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentrazione finale </td></tr><tr><td> Collagenasi di tipo II </td><td> 25 mg </td><td> 0,05% (w / v) </td></tr><tr><td> Proteasi XIV </td><td> 10 mg </td><td> 0,02% (w / v) </td></tr><tr><td> BDM </td><td> 0,025 g </td><td> 5 mM </td></tr><tr><td> Carnitina </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> La taurina </td><td> 0,031 g </td><td> 5 mM </td></tr><tr><td> Acido glutammico </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> 0,1 M CaCl 2 </td><td> 12,5 uL </td><td> 25 uM </td></tr><tr><td> Tampone A </td><td></td><td> Riempire a 50 ml </td></tr></tbody></table> Tabella 4: Ricetta soluzione per 25X BDM / Taurina / BSA (B / T / B) <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentrazione finale </td></tr><tr><td> BDM </td><td> 0,076 g </td><td> 125 mM </td></tr><tr><td> La taurina </td><td> 0,094 g </td><td> 125 mM </td></tr><tr><td> BSA </td><td></td><td> 0,1% (w / v) </td></tr><tr><td> Tampone A </td><td></td><td> Riempire a 6 ml </td></tr></tbody></table> Tabella 5: ricetta per la soluzione tampone B e tamponi di lavaggio <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Buffer: </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> Aggiungere: </td><td></td><td></td><td></td><td></td></tr><tr><td> 0,5 M CaCl 2 </td><td> 0,4 mL </td><td> 0,1 mL </td><td> 0,125 ml </td><td> 0,15 ml </td></tr><tr><td> Concentrazione finale di CaCl 2 </td><td> 1 mM </td><td> 1 mM </td><td> 1,25 mm </td><td> 1,5 mM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 ml </td><td> 2 ml </td><td> 2 ml </td></tr><tr><td> Buffer Un volume di finale </td><td> 200 mL </td><td> 50 mL </td><td> 50 mL </td><td> 50 mL </td></tr></tbody></table> Tabella 6: Ricetta soluzione per Media Culturing <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X siero senza Media (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> Concentrazione finale </td></tr><tr><td> (Dopo diluizione) </td></tr><tr><td> Carnitina </td><td> 0,099 g </td><td> 10 mM (5 mm) </td></tr><tr><td> La taurina </td><td> 0,063 g </td><td> 10 mM (5 mm) </td></tr><tr><td> Creatina </td><td> 0,075 g </td><td> 10 mM (5 mm) </td></tr><tr><td> Antibiotica / antimicotica </td><td> 0,5 ml </td><td> 1% (0,5%) (v / v) </td></tr><tr><td> 199 mezzi di comunicazione </td><td> 45 ml </td><td> Riempire a 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentrazione finale </td></tr><tr><td> 2X SFM </td><td> 25 mL </td><td> 1X </td></tr><tr><td> 199 mezzi di comunicazione </td><td></td><td> Riempire a 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X media del 5% Siero </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentrazione finale </td></tr><tr><td> 2X SFM </td><td> 25 mL </td><td> 1X </td></tr><tr><td> FBS </td><td> 2,5 ml </td><td> 5% (v / v) </td></tr><tr><td> 199 mezzi di comunicazione </td><td></td><td> Riempire a 50 mL </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

Un passo fondamentale è la velocità con cui è appeso il cuore isolato sul sistema di perfusione. La durata del periodo di digestione enzimatica può essere un po &#39;diverso per ogni ratto. La regolazione dipende da come il cuore diventa morbida dopo il periodo normale di digestione. Il piano di recupero di Ca 2 + dopo la digestione enzima è essenziale per ottenere le cellule sane Ca 2 +-tollerante. Per la cavia, il protocollo è lo stesso, tranne ialuronidasi vien...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
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<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
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<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
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<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
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<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
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<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
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<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
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<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
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<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
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<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
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<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
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<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
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primary culture of adult rat heart myocytes

Coltura primaria adulto cellule del cuore roditori sono un modello di sistema importante per la ricerca cardiovascolare. Tuttavia, la creazione di robusti, miociti vitali adulto colto può essere tecnicamente impegnativo, fattore limitante per molti ricercatori. Qui abbiamo descritto un protocollo per ottenere un elevato rendimento di miociti adulti cuore di ratto che rimangono vitali in coltura per diversi giorni. Il cuore è isolato e perfuso con collagenasi e proteasi in basso Ca<sup&gt; 2 +</sup&gt; Condizioni di recuperare singoli miociti. Ca<sup&gt; 2 +</sup&gt;-Tolerant cellule sono ottenute da un aumento graduale in extracellulare Ca<sup&gt; 2 +</supConcentrazione&gt; in tre successive fasi di lavaggio. Le cellule sono filtrati, risospeso in terreno di coltura, e placcato sulla scivola laminina rivestito. Miociti in coltura ottenuti usando questo protocollo sono vitali per un massimo di quattro giorni e sono adatti per la maggior parte degli esperimenti tra cui elettrofisiologia, biochimica, biologia molecolare e di imaging.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

In questo articolo, abbiamo descritto un modo tipico per isolare e cultura miociti adulti cuore di ratto. Collagenasi e proteasi sono utilizzati da digerire e isolare singoli miociti. Miociti colto seguire questo protocollo soddisfare le esigenze più sperimentare.

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 ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.2K Views.  Columbia University. In questo articolo, abbiamo descritto un modo tipico per isolare e cultura miociti adulti cuore di ratto. Collagenasi e proteasi sono utilizzati da digerire e isolare singoli miociti. Miociti colto seguire questo protocollo soddisfare le esigenze più sperimentare.

Video: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Cultura organotipica di Retina Coniglio adulti

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Ricerca

Biologia

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

L&#39;impianto di ingegneria tessutale nel Cuore Rat

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

L&#39;isolamento e la manipolazione genetica di adulti miociti cardiaci per confocale Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Stabilire colture primarie di fibroblasti adulti dai roditori

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Preparazione del Cuore Rat altamente Accoppiato mitocondri

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Metodi per l&#39;isolamento, la cultura, e la caratterizzazione funzionale di seno-atriale Nodo miociti da topi adulti

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Isolamento simultaneo di cardiomiociti di alta qualità, cellule endoteliali e fibroblasti da un cuore di ratto adulto

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

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 ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Colture primarie di cellule corticosurrenali ratto e saggi di Steroidogenic funzioni

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...