Questa ricerca è stata sostenuta in parte dalla American Heart Association concedere 0950118G.to MC-W.

1. Preparazione biglie magnetiche anti-PECAM-1 anticorpo-coniugato (Dynabeads) <ol><li> Preparare 0,1% BSA in PBS mescolando 50 mg in 50 ml di BSA PBS usando un vortice fino a BSA si dissolve. Sterilizzare per filtrazione attraverso filtro 0,22 micron siringa. Conservare a 4 ° C. </li><li> Nel cofano TC, trasferire 200 ul di ben risospeso pecora anti-topo IgG Dynabeads in una provetta Eppendorf da 1,5 ml e lavare le perle: il tubo di Monte MPC e lasciate riposare per circa 1 minuto per consentire le perle di sedimento. Aspirare il surnatante, rimuovere il tubo dal MPC e risospendere le sfere in 1 ml di sterile 0,1% BSA / PBS. Pipetta su e giù per risospendere le sfere. </li><li> Ripetere il lavaggio altre tre volte, per un totale di quattro lavaggi. </li><li> Rimuovere la provetta dal MPC e risospendere le sfere in 500 ml di 0,1% BSA / PBS. </li><li> Aggiungere 10 microlitri di ratto anti-topo PECAM-1 (CD-31) anticorpi al tubo. </li><li> Caduta durante la notte a 4 ° C nella stanza fredda o 2 ore a temperatura ambiente. </li><li> Lavare le perline con sterile 0,1% BSA / PBS come descritto al punto 1,2 quattro volte. </li><li> Rimuovere la provetta dal MPC e risospendere le sfere in 200 l 0,1% BSA / PBS. Conservare anti-PECAM-1 anticorpo-coniugato Dynabeads a 4 ° C per un massimo di 1 mese. </li></ol> 2. Isolare topo le cellule endoteliali polmonari di topi neonati Prima di procedere con il protocollo di purificazione del tessuto, IACUC approvazione del Comitato delle procedura è necessaria. <ol><li> Preparare la seguente: <ul><li> 15 ml di 1 mg / ml C / D soluzione in DMEM. Filtro con siringa da 0,22 micron filtro e calda a 37 ° C. </li><li> Tubo da 50 ml con 15 ml di DMEM, memorizzare sul ghiaccio </li><li> Isolamento Media (IM) contenente 20% FBS e 1 penicillina / streptomicina in DMEM </li><li> 2% di gelatina. Mix 2 g di pelle bovina gelatina con 100 ml di acqua gg, autoclave 15 minuti e conservare a 4 ° C. Calda a 37 ° C per liquefare prima del rivestimento fiaschi TC. </li></ul></li><li> Anestetizzare tre cuccioli di 6-8 giorno di vita con una iniezione IP di ketamina (100 mg / kg) e xilazina (10 mg / kg). Controllare l&#39;efficacia di anestesia e sezionare gli animali uno per uno, come segue: cuccioli Pin a bordo, bagnare la pelle con il 70% etanolo e togliere la pelle dalla zona del torace con le forbici dissezione sterile. Accise la gabbia toracica per esporre polmoni. </li><li> Asetticamente accise lobi polmonari individuale, facendo attenzione a non sezionare i bronchi e visibile qualsiasi tessuto connettivo intorno ai polmoni. Unire tutti polmoni ghiacciata DMEM. Eutanasia dei cuccioli. </li><li> Lavorare nel cofano TC, rimuovere lobi polmonari da DMEM, posto in un piatto da mm 100 TC e aspirare l&#39;eccesso DMEM. </li><li> Usando forbici sterili, tritare finemente il tessuto tagliando ~ 100 volte. Trasferimento a 15 ml caldo C / D soluzione per la digestione enzimatica. Incubare 45 minuti a 37 ° C su un rotatore. </li><li> Nel cofano TC, aspirare la sospensione tessuto digerito in una siringa da 20 ml con 14 g grumi cannula collegata e triturare in una sospensione singola cella, almeno 12 volte. </li><li> Passare la sospensione dei tessuti attraverso un filtro a 70 micron di cellule e lavare il filtro con 15 ml di IM per fermare la digestione. </li><li> Pellet sospensione cellulare mediante centrifugazione a 400x g per 5 minuti. </li><li> Aspirare il surnatante e risospendere pellet in 3 ml 0,1% BSA / PBS. </li><li> Trasferimento sospensione cellulare a 5 ml a fondo rotondo in polistirene e aggiungere 22,5 microlitri anti-PECAM-1 anticorpo-coniugato Dynabeads. Tumble a temperatura ambiente per 12 minuti. </li><li> Un cappotto T75 pallone con 2 ml al 2% di gelatina e aspirare l&#39;eccesso di gelatina. Lasciare asciugare gelatina all&#39;interno della cappa TC per circa 30 minuti prima di cellule placcatura. </li><li> Dopo l&#39;incubazione tallone è completo (passo 2.10), divisi equamente sospensione cellulare in tre provette Eppendorf e montata sul MPC. </li><li> Quando le microsfere sono sedimentate con il MPC (~ 1 min), raccogliere il supernatante, rimuovere tubo da MPC, lavare perline con ~ 1 ml 0,1% BSA / PBS, e poi rimontare sul MPC. </li><li> Ripetere il lavaggio quattro volte (totale 5 lavaggi) </li><li> Perline Risospendere in 1 ml di VascuLife con EnGS-Mv Vita Fattori Kit e 1x penicillina / streptomicina (VL) per tubo, mescolando bene. </li><li> Piastra su un preverniciato T75 pallone e portare il volume totale in ogni pallone a 10 ml con VL. </li><li> Cambiare i media il giorno successivo, 1 giornata piena di crescita, e quindi modificare la metà dei mezzi di comunicazione a giorni alterni. Quando le cellule sono&gt; 70-80% confluenti o più (di solito dopo 3-4 giorni di cultura), possono essere ordinati con anti-ICAM-2 Dynabeads. </li></ol> 3. Preparazione anti-ICAM-2 anticorpo-coniugato Dynabeads <ol><li> Dynabeads anti-ICAM-2 anticorpo-coniugato vengono utilizzati per purificare ulteriormente le cellule che sono state selezionate con anti-PECAM-1 anticorpo-coniugato Dynabeads e colto fino confluenti. Questa procedura viene eseguita come descritto per Dynabeads anti-PECAM-1 anticorpo-coniugato (1, sopra), tranne che anti-topo ICAM-2 (CD-102) viene usato al posto degli anticorpi anti-PECAM-1 anticorpi. </li><li> Dynabeads anti-ICAM-2 anticorpo-coniugato può essere conservato a 4 ° C per un massimoa 1 mese. </li></ol> 4. Ordinamento cellule polmonari endoteliali Mouse con anti-ICAM-2 Dynabeads <ol><li> Cappotto T75 cultura fiasco del tessuto con il 2% di gelatina. </li><li> Aspirare il supporto dal confluenti T75 pallone con le cellule e lavare con 8 ml di PBS. </li><li> Aspirare il PBS, aggiungere 2 ml 0,05% tripsina / EDTA e lasciare che le cellule staccare (circa 5 minuti). Toccare fiaschi leggermente per aiutare distacco. </li><li> Quando le cellule sono staccati, aggiungere 2 ml IM di inibire le cellule tripsina e raschiare di staccare tutte le cellule rimanenti aderente. Trasferimento sospensione cellulare in una provetta da 15 ml e spin down per 5 min a 400x g. </li><li> Aspirare i media e risospendere pellet di cellule in 2 ml 0,1% BSA / PBS. </li><li> Trasferire in un polistirolo da 5 ml a fondo rotondo tubo e aggiungere 10 microlitri anti-ICAM-2 Dynabeads. Biancheria per 12 minuti a temperatura ambiente. </li><li> Split sospensione cellulare in due provette Eppendorf da 1,5 ml e montata sul MPC. </li><li> Aspirare il surnatante dopo perline hanno aderito per un minuto. Rimuovere i tubi da MPC e risospendere ogni tubo in 1 ml 0,1% BSA / PBS. Rimontare il MPC. </li><li> Ripetere il lavaggio quattro volte (totale di 5 lavaggi). </li><li> Risospendere ogni provetta contenente 1 ml VL ed eseguire un conteggio delle cellule. </li><li> Cellule piastra a 250-350K cellule per T25 pallone e / o 750K-1 milioni di cellule per T75 pallone. </li><li> Portare il volume a 5 VL ml in fiasche T25 e T75 in 10 ml fiaschi. </li><li> Cambiare i media a giorni alterni. Le celle possono essere utilizzati per esperimenti quando la cultura raggiunge circa l&#39;80% confluenza, di solito in 2-3 giorni. </li></ol> 5. Rappresentante Risultati In genere, dopo 6-7 giorni dalla preparazione iniziale delle cellule, siamo in grado di ottenere circa 1,2-1,5 x 10 6 cellule endoteliali polmonari da tre 6-8 cuccioli giorno di vita. Cellule visualizzazione tipica morfologia &quot;ciottoli&quot; al microscopio ottico e mostrare VE-caderina (CD144) colorazione a giunzioni cellula-cellula, che è caratteristico per le cellule endoteliali (Figura 1). Maggioranza di esprimere MLECs VE-caderina e VEGFR2 come dimostrato da analisi FACS (Figura 2). Di solito, li usiamo per esperimenti entro due settimane dopo l&#39;isolamento iniziale MLEC. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="Figura 1" /> Figura 1. Analisi al microscopio di confluenti monostrato MLEC. (A) immagine microscopia evidenzia morfologia ciottoli di cellule in coltura tipica per le cellule endoteliali. Uniforme strutture rotondo situato nella zona perinucleare di molte cellule sono le sfere magnetiche utilizzate per l&#39;isolamento MLEC. (B) endoteliali specifici VE-caderina è localizzato presso la cellula-cellula incroci, come indicato usando la microscopia confocale. Bar è rappresentativo di 20μm. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="Figura 2" /> Figura 2 analisi FACS di MLECs colto etichettati con anticorpi specifici per l&#39;endotelio-specifici marcatori:. VE-caderina (CD144) o VEGFR2, come accusato, seguita da ficoeritrina anticorpo secondario coniugato, conferma l&#39;identità di MLECs endoteliali isolate. Linea rossa indica isotipo-specifici di controllo, la linea verde indica anti-VE-caderina o anti-VEGFR2 - specifica-IgG.

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, 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=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+vascular+endothelial+cadherin+(7B4/cadherin-5),+an+endothelium-specific+cadherin.">Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.</a> Arterioscler Thromb Vasc Biol. 15, 1229-1239 (1995).</li><li>Zachary, I., Gliki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+transduction+mechanisms+mediating+biological+actions+of+the+vascular+endothelial+growth+factor+family.">Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family.</a> Cardiovasc Res. 49, 568-581 (2001).</li><li>Diglio, C. A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. E., Gale, D., White, G. C., Vansluys, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+angiogenesis,+endothelial+migration,+proliferation,+and+MAPK+signaling+in+Rap1b-deficient+mice.">Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice.</a> Blood. 111, 2647-2656 (2008).</li></ol>

Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal Medical College of Wisconsin IACUC Comitato ai sensi del protocollo AUA # 00001206.

Microvascolari EC hanno dimostrato di essere un modello utile in molte aree della biologia vascolare e si crede di essere più fisiologicamente rilevanti per lo studio (angiogenesi ad esempio) che ampiamente studiato HUVECs 3. In precedenza, è stato riportato che microvascolare EC possono essere ottenute da reni, cuore, tessuto adiposo della pelle, della retina, del cervello, i gliomi, ma anche polmone 2, 4-7, 10, 11. Tuttavia, un metodo coerente e affidabile per l&#39;isolamento di microvascolare ECS è ancora necessaria. La procedura qui presentata è una modifica di un protocollo precedentemente pubblicato 2; si differenzia dalla maggior parte delle procedure pubblicate in quanto utilizza cuccioli invece di animali adulti. Questo è fondamentale per il successo isolamento di cellule da giovani animali hanno un potenziale di proliferazione maggiore e culture deriva dal loro tessuti tendono a produrre un numero maggiore di cellule. Una settimana di vita gli animali sembrano essere ottimali, ma più giovani topi (giorno-vecchio 4) può anche essere usato. Inoltre, i numeri più alti o più bassi di cuccioli può essere utilizzata, se necessario, in quanto siamo riusciti a isolare MLECs da un cucciolo. Tuttavia, mentre la scala verso l&#39;alto o verso il basso il processo di isolamento, placcatura densità cellulare deve rimanere invariato, in quanto questo è anche fondamentale per la proliferazione cellulare. Il 2-step del processo di purificazione MLECs utilizzando biglie magnetiche coniugate prima anti-PECAM-1 e di anti-ICAM-2 anticorpi è molto più efficiente ad ottenere puro culture CE rispetto alla precedentemente descritte passo-passo le procedure di purificazione. Questo protocollo elimina la necessità di utilizzare tecniche manuali molto laborioso, centrifugazione gradiente e FACS meno efficiente di smistamento per le cellule scartando contaminanti, come i fibroblasti, cellule del sangue e cellule muscolari lisce. La popolazione risultante MLEC può essere utilizzato per l&#39;analisi in vitro delle risposte angiogenici, permeabilità vascolare e la trasmigrazione dei leucociti, la guarigione della ferita e l&#39;analisi biochimica delle vie di segnalazione. Se necessario, MLECs può essere placcato su superfici diverse, come coprioggetto fibronectina o gelatina rivestite di immunofluorescenza o array di elettrodi per la trans-endoteliale resistenza (TER) misurazioni. Risultati ottenuti possono essere direttamente confrontati con i fenotipi osservate in vivo, che è particolarmente importante nel contesto del crescente numero di eliminazione diretta e le linee di topi transgenici. Il successo di questo metodo per l&#39;analisi in vitro dei meccanismi cellulari alla base difetti osservate in vivo, è già stato presentato 12.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of pulmonary endothelial cells from neonatal mice

Le cellule endoteliali forniscono un utile modello di ricerca in molte aree della biologia vascolare. Fin dal suo primo isolamento 1, cellule endoteliali della vena ombelicale (HUVECs) hanno dimostrato di essere comodo, facile da ottenere e cultura e, quindi, sono le cellule più ampiamente studiato endoteliali. Tuttavia, per la ricerca si è focalizzata sui processi come gli altri angiogenesi, permeabilità o molti, cellule endoteliali (EC) sono un modello molto più fisiologicamente rilevanti per lo studio 2. Inoltre, EC isolate da topi knockout per fornire un utile strumento per l&#39;analisi ex-vivo di funzione della proteina. Diversi approcci per isolare e microvascolare cultura EC di diversa provenienza sono stati segnalati fino ad oggi 3-7, ma consistente isolamento e la cultura della pura ECS è ancora un grave problema tecnico in molti laboratori. Qui, forniamo un passo-passo protocollo su un metodo affidabile e relativamente semplice di isolare e coltura delle cellule endoteliali del polmone del mouse (MLECs). In questo approccio, tessuto polmonare ottenuti da 6 - a 8 giorni di cuccioli di età è prima tagliato a pezzi, digerito con collagenasi / dispasi (C / D) la soluzione e disperse meccanicamente in cella singola sospensione. MLECS sono purificati dalla sospensione cellulare tramite selezione positiva con l&#39;anti-PECAM-1 coniugato a Dynabeads utilizzando un concentratore di particelle magnetiche (MPC). Tali cellule purificate sono coltivati ​​su gelatina rivestita coltura tissutale (TC) piatti fino a diventare confluenti. A quel punto, le cellule sono ulteriormente purificato con Dynabeads accoppiato ad anti-ICAM-2 anticorpi. MLECs ottenuti con questo protocollo mostra un fenotipo ciottoli, come visualizzato in contrasto di fase microscopia ottica, e il loro fenotipo endoteliale è stata confermata con analisi FACS con anti-VE-caderina 8 e anti-VEGFR2 9 anticorpi e colorazione di immunofluorescenza di VE-caderina. Nelle nostre mani, questa procedura in due fasi isolamento in modo coerente e affidabile produce una popolazione pura di MLECs, che può essere ulteriormente coltivate. Questo metodo permetterà ai ricercatori di sfruttare il crescente numero di topi transgenici e knockout per correlare direttamente studi in vivo con i risultati di esperimenti in vitro eseguiti su MLECs isolato, contribuendo così a svelare i meccanismi molecolari del fenotipo vascolare osservata in vivo.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Qui, descriviamo un protocollo per l&#39;isolamento e la coltura di cellule murine endoteliali polmonari. Questo metodo comprende meccanico e dissociazione enzimatica tessuto polmonare e una 2-step processo di purificazione con anti-PECAM-1 e anti-ICAM-2 anticorpi coniugati a sfere magnetiche, che produce una popolazione pura di cellule endoteliali di origine per lo più microvascolare.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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JoVE Journal

Biology

46.9K Views.  BloodCenter of Wisconsin. Qui, descriviamo un protocollo per l'isolamento e la coltura di cellule murine endoteliali polmonari. Questo metodo comprende meccanico e dissociazione enzimatica tessuto polmonare e una 2-step processo di purificazione con anti-PECAM-1 e anti-ICAM-2 anticorpi coniugati a sfere magnetiche, che produce una popolazione pura di cellule endoteliali di origine per lo più microvascolare.

Video: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolamento di cellule endoteliali valvolare

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Ricerca

Biologia

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolamento e Cultura della neonatali mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento della embrionali ventricolari cellule endoteliali

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolamento di polmonare cellule muscolari lisce di topi neonati

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Generazione e Cultura Sangue escrescenza endoteliali cellule da sangue periferico umano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento di cellule di topo coronarica endoteliali

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

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 ...