Questo lavoro è stato sostenuto dalla sovvenzione da parte del National Institutes of Health (HL115578 ad A. Makino).

La ricerca sui topi è stato approvato dal Comitato Istituzionale Animal Care e Usa (IACUC) della University of Arizona ed è stata eseguita secondo il National Institute of Health (NIH) le linee guida. NOTA: Le sezioni 1, 2 e 3 devono essere effettuate il giorno prima l&#39;esperimento come impostazione. 1. Soluzioni Preparazione <ol><li> buffer di CD31 (50 ml) <ol><li> Aggiungere 50 ml 1 × Tampone fosfato salino (PBS) ad un tubo 50 ml. Pesare 0,05 g albumina sierica bovina (BSA) e 0,037 g di acido etilendiamminotetraacetico (EDTA) e aggiungere al tubo da 50 ml e mettere il tubo di ruotare per circa 1 ora fino a quando si dissolve in polvere. Regolare il pH a 7,4, filtrare attraverso un filtro da 0,2 micron in un nuovo tubo da 50 ml, e conservare a 4 ° C fino al momento dell&#39;uso. </li></ol></li><li> Soluzione salina bilanciata di Hank (HBSS) con HEPES (500 ml) <ol><li> Aggiungere 1,1915 g di HEPES per fare 10 mM HEPES in soluzione HBSS e mescolare bene. Misurare il pH e regolare a 7,4. Filtro e conservare a 4˚C. </li></ol></li><li> La soluzione di 1x Krebs (1 L) <ol><li> Aggiungere 100 ml di soluzione di 10 × di Krebs [Tabella 1] per raddoppiare acqua distillata in un pallone da 1 L. Pesare 2,1 g di sodio bicarbonato e 2 g di D-glucosio e aggiungere al pallone. Mescolare e regolare il volume totale di 1 L. </li><li> Soluzione Bubble con 95% O 2/5% CO 2 gas per 30 min e filtro soluzione all&#39;interno della cappa. Conservare a 4 ° C e tenere in ghiaccio durante l&#39;esperimento. </li></ol></li></ol> 2. Preparare le biglie magnetiche <ol><li> Aggiungere 1 ml di tampone CD31 in ogni provetta 1,5 ml (un tubo per campione). Mescolare le perline IgG anti-ratto pecore e aggiungere la giusta quantità di ogni campione [Tabella 1]. Agitare vigorosamente le provette 30 volte e metterli nella piastra magnetica per 1 min di agitazione. Aprire i tubi, mentre sul piatto e scaricare le soluzioni in un secchio. </li><li> Aggiungere 1 ml di tampone CD31 in ogni provetta. Aggiungere 2 ml Rat anti-topo CD31 anti-corpo ad ogni provetta e ruotare i tubi O / N a 4 ° C. </li></ol> 3. autoclave <ol><li> Autoclavare tutti gli strumenti di chirurgia e punte per pipette. Tagliare le estremità delle punte per pipette in modo che non ci sono punte con 4 diametri diversi, dal più largo a stretto. diametri approssimativi per le punte sono di 3 mm, 2,5 mm, 2 mm e di 1,5 mm, rispettivamente. Tagliare le punte e autoclave il giorno prima con 30 min di sterilizzazione ad una temperatura di 121 ° C. NOTA: Sezioni 4 - 11 dovrebbero essere eseguite il giorno dell&#39;esperimento. </li></ol> 4. Soluzioni Preparazione <ol><li> Preparare il tampone di lavaggio contenente il 2% di ferro-fortificato Calf Serum (FISC) in M199. Per l&#39;imaging, preparare 30 ml / mouse. </li><li> Preparare la soluzione enzimatica (10 ml / campione). Pesare 0,6 unità / ml di dispasi e 1 mg / ml di Collagenasi tipo II in un tubo da 50 ml. Aggiungere M199 e ruotare il tubo a 4 ° C per 15 min. Filtrare 0,2 micron filtro in un nuovo tubo da 50 ml inside il cofano e mantenere a 4 ° C fino al momento dell&#39;uso. </li><li> Preparare HBSS / HEPES con eparina. Aggiungere 10 ml di HBSS con 10 mM HEPES in 15 ml tubo. Aggiungere 100 ml di eparina al tubo e mantenere a 4 ° C fino all&#39;utilizzo. </li></ol> 5. Lavare le perline <ol><li> Estrarre i tubi con perline del rotatore a 4 ° C e portare alla cappa. Posizionare i tubi nella piastra magnetica e agitare per 1 min. Dump la soluzione tampone e aggiungere CD31 1 ml in ogni provetta. </li><li> Prendere i tubi fuori dalla piastra magnetica e agitare vigorosamente 30 volte. Mettere i tubi indietro nella piastra magnetica e ripetere per un totale di 3 lavaggi. Dopo il completamento, mettere i tubi di nuovo a 4C e mantenere la rotazione fino al momento dell&#39;uso. </li></ol> 6. Dissection <ol><li> Iniettare topi per via intraperitoneale con 0,1 ml di eparina per prevenire la coagulazione del sangue. Attendere 10 minuti, quindi anestetizzare i topi con iniezione intraperitoneale di 0,01 ml di pentobarbital. Controllare se il mouse si senteil dolore da pizzicare il piede. </li><li> Posizionare il mouse su una tavola di polistirolo con la testa e tenere le braccia con perni. Utilizzare pinze e forbici-cut-duri per tagliare la pelle nella zona toracica superiore fino alla gola. </li><li> Effettuare un taglio nel centro della gabbia toracica appena sopra il diaframma. Tagliare l&#39;osso lateralmente e verso l&#39;alto verso la gola per fare un triangolo esponendo il cuore e polmoni. Tagliare l&#39;aorta appena sopra il diaframma. </li><li> Tenere il timo con una pinza senza tirare e usare le forbici per tagliare con attenzione qualsiasi tessuto connettivo per rimuovere polmoni e il cuore insieme. Posizionare i tessuti in un bicchiere con 1 × Kreb di, agitare con una pinza e trasferimento a 6 centimetri piatto con 1 × Kreb di. </li><li> Rimuovere i lobi polmonari con le forbici e pinze. Inserire un catetere sterile 20 G nell&#39;aorta. Lavare il sangue nel sistema circolatorio coronarico con HBSS / eparina. Fare un piccolo taglio nel ventricolo, agitare il cuore e trasferirli in una provetta di M199. Mantenere i tubi in ghiaccio. </li></ol><p<html> class = &quot;jove_title&quot;&gt; 7. Tissue Digestione <ol><li> Trasferire il cuore di un 6 centimetri piatto riempito con 10 ml di soluzione enzimatica e tritare in piccoli pezzi con le forbici. Porre le capsule in incubatore a 37 ° C per 15 min. Agitare materiali digeriti pipettando su e giù 30 volte attraverso una punta con una estremità tagliata per permettere al tessuto di passare attraverso più facile. </li><li> Rimetterlo nel 37 ° C incubatore per altri 15 min. Ripetere l&#39;agitazione per tre volte, ogni volta con punte per pipette da grandi aperture a più stretto e 15 min di incubazione tra ciascuna. </li><li> Dopo l&#39;ultimo 15 minuti di incubazione, prelevare campioni fuori e dentro il cofano. soluzione Pipettare su e giù per 30 volte utilizzando punta della pipetta con l&#39;apertura più stretto e passare attraverso un colino 40 micron cella in un nuovo tubo da 50 ml. </li><li> Lavare il piatto con tampone di lavaggio 10 ml con una pipetta 10 ml e trasferire su colino cella. Mettere 50 ml provette nella centrifuga e centrifugare a 400 g per 10 minuti a RT. </li></ol>TITOLO &quot;&gt; 8. Lavaggio e coniugazione con perline <ol><li> Rimuovere il surnatante senza disturbare il pellet. Lavare le cellule con tampone di lavaggio 5 ml due volte. </li><li> Ottenere una delle precedentemente preparato provette da 1,5 ml con perline e lavorare su un tubo alla volta. Mettere una provetta da 1,5 ml nella piastra magnetica, agitare per 3 volte e aprirlo. </li><li> Dopo l&#39;ultima centrifugazione, rimuovere il più surnatante possibile senza disturbare il pellet. Dissociare i grumi di cellule muovendo vigorosamente. </li><li> Aggiungere tampone di lavaggio 1 ml al tubo 50 ml e mescolare le cellule con una pipetta 1 ml. Dump la soluzione dalla provetta da 1,5 ml e aggiungere 1 ml di sospensione cellulare dal tubo 50 ml ai talloni. Flick i tubi 30 volte poi ruotare a 4 ° C per 30 minuti. </li></ol> 9. Preparazione della coltura cellulare / Imaging <ol><li> Preparare le camere per la placcatura cellule. Aggiungere la soluzione di gelatina (5% w / v, 300 microlitri) alla camera bene per rivestire la superficie e incubare per 30 min a 37 ° C. Dopo l&#39;incubazione, remgelatina ove e asciugare la superficie. </li><li> Estrarre i tubi con perline / cellule dopo 30 minuti di rotazione e mettere nel piatto magnetico all&#39;interno della cappa. Agitare per 2 minuti, discarica e aggiungere tampone di lavaggio 1 ml. </li><li> Prendere tubi dalla piastra, agitare energicamente 30 volte poi flick 30 volte. Metteteli nel piastra magnetica e agitare per 1 min. Ripetere 4 volte per un totale di 5 lavaggi, ma con 1 min agitando sulla piastra. </li><li> Dopo l&#39;ultimo lavaggio, estrarre un tubo e lavorare su ciascun tubo uno alla volta. Agitare 30 volte poi scorri 30 volte. Mettere sul piatto magnetico e agitare per 1 min. Dump e aggiungere 1 ml di 20% dei media IFCS CE (pre-riscaldato a 37 ° C). </li><li> Agitare vigorosamente 30 volte poi flick 30 volte. Regolare pipetta a 500 microlitri e pipettare su e giù per 10 volte. Trasferire 500 microlitri di ciascuna camera. </li><li> Mettere a 37 ° C incubatore e lasciare O / N. Il giorno dopo, lavare le cellule con il 10% dei media FBS CE. </li></ol> 10. Preparazione per Western Blot (WB) Campioni <ol><li> Takuna e le provette con perline / cellule dopo 1 ora di rotazione. Metteteli nella piastra magnetica e agitare per 2 min. Dump surnatante e aggiungere 1 ml HBSS freddo. Agitare vigorosamente 30 volte poi flick 30 volte. Ripetere due volte per 3 lavaggi, ma con 1 min agitando sulla piastra. </li><li> Dopo l&#39;ultimo lavaggio, mettere i tubi sulla piastra magnetica e agitare per 1 min. Rimuovere tampone utilizzando pipetta e lisare le cellule per WB utilizzando un metodo descritto in precedenza 11,12. lisato raccolte di solito contiene circa 30 mg di proteina. </li></ol> 11. Imaging <ol><li> Lavare le cellule 3 volte con supporti, con volume finale di 250 microlitri di una camera. Aggiungere 1,5 ml di Dil-acLDL alle cellule. Da questo punto, mantenere le cellule nel buio. </li><li> Incubare per 3,5 ore a 37 ° C. Aggiungere 1,5 ml di Hoechst, mescolare bene e incubare per 30 minuti a 37 celle ˚C.Wash con PBS e fissare con 4% paraformaldeide (PFA) per 20 minuti a temperatura ambiente. </li><li> Lavare due volte con PBS e bloccare con il 5% di BSA-PBS per 30 min a temperatura ambiente. Lavare due volte con 1% BSA-PBS. Diluire BS-lectina-FITC usando 1% BSA-PBS ad una concentrazione di 2 mg / ml e aggiungere 300 microlitri di ciascuna camera. </li><li> Mettere la camera sul agitatore orbitale a temperatura ambiente per 30 min. Lavare con PBS 3 volte. </li><li> Visualizzare le cellule sotto il microscopio a fluorescenza con il tempo di esposizione di 200 msec per FITC (colorazione lectina, un marker CE, di eccitazione / emissione di 488 nm / 519 nm), 30 msec per DAPI (Hoechst, una colorazione, di eccitazione / emissione nucleare a 358 nm / 461 nm), e 300 msec per TRITC (acLDL colorazione, un marker CE, di eccitazione / emissione a 557 nm / 576 nm). Acquisire le immagini utilizzando una lente obiettivo 20X. </li></ol>

<ol><li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell+functions%5BTitle%5D)+AND+%22J+Cell+Physiol%22%5BJournal%5D)">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li>
<li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+in+focus%3A+endothelial+cell%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li>
<li>Cines, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li>
<li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+function+and+dysfunction%3A+testing+and+clinical+relevance%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li>
<li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li>
<li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li>
<li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+endothelial+cells+from+murine+tissue%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li>
<li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+endothelial+cells+from+fresh+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li>
<li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li>
<li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li>
<li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li>
<li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VDAC%3A+old+protein+with+new+roles+in+diabetes%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li>
</ol>

The authors have nothing to disclose.

Le cellule endoteliali più utilizzati nella ricerca sono cellule umane endoteliali della vena ombelicale (HUVEC) a causa della loro praticità e facilità di coltura. Tuttavia, molte ricerche richiede la disponibilità di un modello fisiologico raggiunto con l&#39;isolamento delle cellule endoteliali da altri organi specifici 10. Le cellule endoteliali formano una popolazione molto minore di cellule nel tessuto cardiaco; il loro isolamento e la coltura grado di dimostrare di essere molto difficile. <p cl...

modelli murini di varie malattie cardiovascolari e disturbi metabolici portano cambiamenti fisiologici e molecolari che sono simili a quelli riscontrati in pazienti. Inoltre, l&#39;alterazione genetica dei topi è un potente strumento che ci permette di indagare il ruolo patogenetico di geni specifici nello sviluppo e nella progressione delle malattie. Al giorno d&#39;oggi,-specifica delle cellule di tipo gene-knock-in o topi -out si generino facilmente in molti laboratori e la misurazione di mRNA e livelli di proteine ​​in specifici tipi di cellule è il primo passo per determinare se i topi sono stati veramente geneticamente modificati. Endotelio è un sottile strato singolo di cellule endoteliali (ECS) che riveste la parete vascolare interno. Le cellule endoteliali giocano un ruolo critico nella regolazione della tensione vascolare, la permeabilità vascolare, e la formazione di nuovi vasi 1,2. La disfunzione endoteliale è un segno distintivo di molte condizioni patologiche e cambiamenti nella funzione endoteliale può portare a diverse automalattie cardiovasco- 3,4. È quindi significativo per studiare la funzione delle cellule endoteliali in condizioni fisiologiche e fisiopatologiche. Ci sono diversi modi per isolare EC 5-8 e il metodo di isolamento deve essere ottimizzato a seconda di quale verranno utilizzati i tessuti e le specie. Questo perché EC da tessuti differenti di un elevato livello di eterogeneità rispetto ai marcatori di superficie e proteica 9. isolamento di successo di cellule endoteliali spesso può essere difficile e richiede un certo grado di formazione e la pratica. Una volta ottenuto, il metodo di isolamento proposto in questo protocollo rivela stabile ed efficiente. L&#39;obiettivo generale di questo metodo è quello di ottenere il mouse coronarica ECS (MCECs) di alta qualità e quantità. Anche se la quantità di cellule raccolte da un cuore è meno di cellule raccolte da altri tessuti mediante altre tecniche, questa tecnica fornisce ancora betqualità ter. La purezza delle cellule endoteliali nella popolazione risultante è superiore al 90%. Così, questa tecnica sarebbe l&#39;ideale per le applicazioni che si basano su cellule endoteliali puramente isolate come l&#39;imaging cellulare.

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

isolation of mouse coronary endothelial cells

Le cellule endoteliali linea la parete interna dei vasi sanguigni e svolgono un ruolo importante nella regolazione del tono vascolare, la permeabilità vascolare, e la formazione di nuovi vasi. La disfunzione delle cellule endoteliali è implicato nello sviluppo e nella progressione di molte malattie cardiovascolari, tra cui cardiopatia ischemica. Per esaminare la funzione e caratterizzazione di cellule endoteliali coronariche, isolamento delle cellule è il primo passo e richiede elevata purezza e quantità di condurre esperimenti successivi. Questo protocollo descrive un metodo efficiente per isolare topo adulto coronarie cellule endoteliali. Il cuore del mouse è sezionato e tritata in piccoli pezzi. Dopo la digestione del cuore utilizzando dispasi e collagenasi II, le cellule vengono lavate e incubate con perline magnetiche che sono coniugate con anticorpi anti-CD31. Le perle con le cellule endoteliali vengono lavati diverse volte e sono pronti per l&#39;uso in varie applicazioni, tra cui l&#39;imaging molecolare e biologica experimenti. Isolamento efficiente produce circa 10 4 cellule per un cuore con oltre il 90% di purezza.

L&#39;isolamento di successo delle cellule endoteliali coronariche da un cuore del mouse si ottiene in genere circa 10 4 cellule con oltre il 90% di purezza con una forma di ciottoli. Per una popolazione pura di cellule endoteliali, si deve usare cautela in tutto il protocollo al fine di garantire un ambiente sterile priva di qualsiasi contaminazione. La comparsa di cellule allungate o allargate cellule all&#39;interno della popolazione indica contaminazione con altri tipi di ...

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

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-of-mouse-coronary-endothelial-cells

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11.2K Views.  University of Arizona, Tucson. 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.

Video: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

Isolamento delle cellule endocartali e coronarie dal muro libero ventricolare del cuore di ratto

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Ricerca

Biologia dello sviluppo

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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 ...

Biologia

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Isolamento di cellule endoteliali microvascolari murino primario del muscolo scheletrico

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neuroscienze

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolamento delle cellule endoteliali polmonari primarie di topo

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunologia e infezioni

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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolamento e caratterizzazione di cellule endoteliali sinusoidali primarie del fegato di topo

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolamento delle cellule endoteliali dal lume delle arterie carotidi di topo per esperimenti multi-omici a singola cellula

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...