This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Isolierung von Aorta von Mäusen Alle hier beschriebenen Verfahren wurden von der Institutional Animal Care und Use Committee von der Wayne State University genehmigt. <ol><li> Setzen Sie die Maus in die Induktionskammer der Anästhesiemaschine. Stellen Sie die Isofluran - Flow auf 4% in Verbindung mit 25% Frischluft und 75% O 2. Anesthetize die Maus durch Inhalation von Isofluran (4% für die Induktion, ± 1,0% für die Wartung) in Verbindung mit 25% Frischluft und 75% O 2 über Induktionskammer durch einen eigenen Bugnase gefolgt. HINWEIS: Anästhesie mit Isofluran kann in 75% Sauerstoff / 25% Luft oder 100% Sauerstoff zugeführt werden. </li><li> Überprüfen Sie die Maus alle 5 Minuten, bis die angemessene Höhe der Anästhesie erreicht ist (Mangel an Rückzug bis Fuß Prise). </li><li> Nehmen Sie die Maus aus der Induktionskammer und weiter Isofluran Inhalation obwohl die spezielle Nasenkegel, die mit dem Anästhesiegerät angeschlossen ist. Schalten Sie dieIsofluran fließen zu 1% Anästhesie aufrechtzuerhalten. </li><li> Während es unter Narkose ist, legen Sie die Maus auf eine Operation Pad, auf dem Rücken positioniert. Verwenden Labor Band, die Glieder zu sichern. </li><li> Verwenden Sie eine Wärmelampe die Maus warm zu halten. Halten Sie die Lampe in einem angemessenen Abstand von der Maus, so dass die Maus nicht überhitzen. </li><li> Sprühen Sie die Brust mit 70% Ethanol. </li><li> Verwenden Sie Dissektion Schere den Bauch von der Mittellinie zu öffnen und die Bauchaorta aussetzen. Öffnen Sie die Brusthöhle und setzen das Herz und die Lunge. </li><li> Schneiden Sie die Bauchaorta in der Mitte mit Dissektion Schere, um das Blut zu lösen. </li><li> Füllen einer 1 ml Spritze (25 G Nadel) mit 1 ml PBS, enthaltend 1,000 U / ml Heparin. Injizieren PBS, enthaltend 1,000 U / ml Heparin zu dem linken Ventrikel und die Aorta perfundieren. Schieben Sie das Herz und die Lunge mit einer Pinzette an den großen Arterien auf die rechte Seite der Mäuse der thorakalen Aorta zu belichten. </li><li> Entfernen Sie schnell die Brustaorta Mikro di mitssection Zange und legte sie in eiskaltem 1x PBS (steril), transportieren dann den Behälter in eine laminare Luftströmung Haube. </li><li> Legen Sie eine 1-ml-Spritze mit 25 G-Nadel in ein Ende der Aorta eingesetzt und sanft die Aorta mit eiskaltem PBS spülen das Blut zu entfernen. </li><li> Verwenden einer Pinzette Mikrodissektion so viel des anhänge Fettgewebe und kleine seitliche Gefäße wie möglich zu entfernen. </li><li> Unmittelbar nach der Aorta übertragen Wachstumsmedium an Endothelzellen. </li><li> Schneiden Sie die Aorta in 1 mm Ringe ein steriles Skalpell verwendet wird. Ernte etwa 8 - 10 Ringe pro Aorta. </li><li> Öffnen Sie jeden Aorten-Ring ein Paar von Mikro-Dissektion Schere. </li></ol> 2. Seed die Aorten-Segmente auf Matrix <ol><li> Wenn nicht in Gebrauch, halten den Wachstumsfaktor reduzierte Matrix in 20 ° C Erstarrungs zu verhindern. Setzen Sie den Wachstumsfaktor reduzierte Matrix in 4 ° C mindestens über Nacht ein komplettes Tauwetter zu ermöglichen. </li><li> Vorkühlung eine 6-Well-Platte und Pipettenspitzen bis -20 ° C für mindestens10 Minuten. Legen Sie die 6-Well-Platte auf Eis und Mantel eine Vertiefung der Platte mit 1 ml Matrix ohne Luftblasen einzuführen. Die Platte wird in einem 37 ° C Inkubator für 20 Minuten die Matrix zu ermöglichen, sich zu verfestigen. </li><li> Implantieren Sie die Aorten-Stücke auf die verfestigte Matrix mit sterilen Mikro-Dissektion Pinzette. Legen Sie die Stücke Lumen-Seite nach unten auf die Matrix ohne das Endothel zu berühren. Platzieren von 3 bis 4 aortic Segmente nahe beieinander auf der Matrix. </li><li> In gerade genug endothelialen Zellwachstumsmedium zu halten Segmente nass (~ 200 ul). </li><li> Inkubieren der Platte bei 37 ° C unter 5% CO 2 für 4 bis 6 h. Am Ende des Tages, fügen Sie einfach genug Medium die Aorten-Segmente abzudecken. </li><li> Beachten Sie die Aorten-Segment in regelmäßigen Abständen unter einem Phasenkontrastmikroskop sprießen. Überprüfen mittleren Ebene und das Zellwachstum pro Tag. In Medium bei Bedarf abgedeckt Aorten-Segmente zu halten. </li><li> Am 4. Tag, entfernen Sie das Medium und entfernen Sie die Aorten - Segmente fROM mit der Matrix mit einer sterilen Nadel ohne die wachsenden Endothelzellen zu unterbrechen. </li><li> In 2 ml neuen endothelialen Zellwachstumsmedium und damit die Endothelzellen weiterhin auf Matrix 2 zu vermehren - 3 Tage. </li></ol> 3. Erste Passagierung der Maus Aortic Endothelial Cells <ol><li> Mantel ein neuer T12.5 Kolben mit Gelatine (0,1%) für 30 min bei 37 ° C inkubieren. </li><li> Waschen Sie die Matrixplatten vorsichtig mit sterilem PBS 37 ° C. </li><li> In 2 ml neutralen Proteinase (50 U / ml) auf die Matrixplatten und Inkubation bei Raumtemperatur auf Wippschüttler mit gelegentlichem Schütteln. Schauen Sie sich die Zellen unter einem Phasenkontrastmikroskop die Mehrzahl der Zellen, um sicherzustellen, abgelöst werden. </li><li> 2 mL D-Val in die neutrale Proteinase zu inaktivieren. </li><li> Sammeln Sie den Überstand vorsichtig in ein 15 ml Zentrifugenröhrchen. Waschen Sie die Platte mit 2 ml D-Val und sammeln den Überstand. </li><li> Zentrifugieren Sie die Zellsuspension bei 900 g für 5 min bei room Temperatur. </li><li> Resuspendieren des Zellpellets in 4 ml Endothelzellen-Wachstumsmedium und der Platte in dem T12.5 Kolben mit Gelatine beschichtet. Inkubieren der Zellen bei 37 ° C, 5% CO 2 für 2 h. </li><li> Ersetzen Sie das Medium. Inkubieren der Zellen bei 37 ° C, 5% CO 2 , bis sie 85% - 90% konfluent. </li></ol> 4. Passagierung der Maus Aortic Endothelial Cells <ol><li> Mantel zwei neue T12.5 Kolben mit Gelatine (0,1%) für 30 min bei 37 ° C inkubieren. Vorheizen Trypsin-EDTA (0,25% Trypsin, 0,02 EDTA in PBS) und sterile PBS bei 37 ° C für etwa 15 min. </li><li> Waschen Sie die Zellen vorsichtig mit einer sterilen 37 ° C PBS. </li><li> In 0,5 ml Trypsin-EDTA (0,25% Trypsin, 0,02 EDTA in PBS) und Inkubation bei 37 ° C für ca. 1 min oder bis die Mehrzahl der Zellen umdrehen. </li><li> 2 mL endothelialen Zellwachstumsmedium, das die Verdauung zu stoppen. Pipette die Zellsuspension nach oben und unten in den Kolben mehrmals. Verwenden Sie bei Bedarf eine ZelleSchaber um die Zellen zu sammeln. </li><li> Sammeln der Zellsuspension in ein 15 ml Zentrifugenröhrchen. Zentrifugieren Sie die Zellsuspension bei 900 × g für 5 Minuten bei Raumtemperatur. </li><li> Resuspendieren des Zellpellets in 4 ml Endothelzellen-Wachstumsmedium und der Platte in 2 T12.5 Kolben mit Gelatine beschichtet. Inkubieren der Zellen bei 37 ° C, 5% CO 2 für 2 h. </li><li> Ersetzen Sie das Medium. Inkubieren der Zellen bei 37 ° C, 5% CO 2 , bis sie 85% - 90% konfluent. </li><li> Verwenden Sie die Maus Aorten-Endothelzellen nach 2 - 3 Passagen. </li></ol> 5. Charakterisierung der Maus Aortic Endothelial Cells <ol><li> Re-Platte die Zellen. <ol><li> Mantel eine 6-well-Platte mit Gelatine (0,1%) für 30 min bei 37 ° C inkubieren. Spülen Sie die Platte mit PBS. </li><li> Vorheizen Trypsin-EDTA und sterilem PBS auf 37 ° C für etwa 15 min. </li><li> Waschen Sie die Zellen mit steriler PBS 3 mal. In 0,5 ml Trypsin / EDTA zu den Zellen, Inkubation für ca. 1 min bei 37 ° C or, bis die Mehrheit der Zellen umdrehen. </li><li> 2 mL endothelialen Zellwachstumsmedium, das die Verdauung zu stoppen. Pipette die Zellsuspension nach oben und unten in den Kolben mehrmals. Verwenden Sie bei Bedarf einen Zellschaber um die Zellen zu sammeln. </li><li> Sammeln der Zellsuspension in ein 15 ml Zentrifugenröhrchen. Zentrifugieren Sie die Zellsuspension bei 900 × g für 5 Minuten bei Raumtemperatur. </li><li> Überstand verwerfen, wieder die Zellen in endothelialen Zellwachstumsmedium. Samen , die Zellen in 6-Well - Platte , beschichtet mit Gelatine in einer Dichte von 3 x 10 5 / Vertiefung. </li><li> Inkubieren der Zellen über Nacht bei 37 ° C, 5% CO 2 werden die Zellen zu erlauben , auf die Kulturoberfläche zu befestigen. </li></ol></li><li> Dil-Ac-LDL uptaken und Ulex -lectin Bindung. <ol><li> Bewahren Sie die 1,1&#39;-Dioctadecyl-3,3,3 &#39;, 3&#39;- tetramethylindo-Carbocyaninfarbstoffes Perchlorat-markierten acetyliertes LDL (Dil-Ac-LDL) bei -20 ° C. Vor dem Färben, auftauen Dil-Ac-LDL auf Eis. Die Aktie Dil-Ac-LDL solution beträgt 1 mg / mL. Lagern Sie den FITC-markierten Ulex europaeus Agglutinin (Ulex -Lectin, 1 &amp; mgr; g / ml) bei 4 ° C. Die Aktie Ulex -Lectin Lösung beträgt 1 mg / ml. Store Hoechst 33342 bei 4 ° C. Die Aktie Hoechst 33342 Lösung ist 100 ug / ml. </li><li> Fügen Sie 10 &amp; mgr; l von Dil-Ac-LDL-Stammlösung auf 1 ml Kulturmedium in einer Endkonzentration von 10 ug / ml zu erhalten. </li><li> Vor der Färbung, entfernen Sie das alte Medium und waschen Sie die Zellen in jeder Vertiefung mit neuen Medium. </li><li> Nehmen Sie das neue Medium und fügen Sie die Dil-Ac-LDL-Färbung Medium. </li><li> Inkubieren der Zellen bei 37 ° C, 5% CO 2 für 1 h. </li><li> Waschen der Zellen mit sterilem PBS 1x 3 mal. </li><li> 1 ml 10% Formalin, die Zellen zu fixieren. Legen Sie die Platte in Raumtemperatur für etwa 30 min. </li><li> Waschen der Zellen mit sterilem PBS 1x 3 mal. Vermeiden Sie Licht. </li><li> In 1 ml sterile 1x PBS , die 10 &amp; mgr; l von Ulex -Lectin enthält. </li><li> Inkubieren Sie die Zellen bei Raum temperature im Dunkeln für 1 h. </li><li> Entfernen Sie die Ulex-Lektin, indem die Zellen mit steriler PBS 1x 3-mal gewaschen wurde. </li><li> Hinzufügen Hoechst 33342 bis zu einer Endkonzentration von 1 ug / ml, 10 min Inkubieren im Dunkeln. </li><li> Sehen Sie sich die Fluoreszenz von Dil-Ac-LDL (rot, Anregungswellenlänge von 576 nm), Ulex -Lectin (grün, Anregungswellenlänge von 519 nm) und Hoechst (blau, Anregungswellenlänge von 361 nm) mit einem invertierten Fluoreszenzmikroskop. </li></ol></li><li> Fluoreszenzfärbung für CD31, VEGFR2, eNOS, VE-Cadherin und Calponin. <ol><li> Store FITC-konjugiertem anti-Maus-CD31-Antikörper, Antikörper eNOS, VE-Cadherin-Antikörper FITC-konjugiertem anti-Kaninchen-IgG bei 4 ° C im Dunkeln. Store VEGFR2 Antikörper in -20 ° C. </li><li> Waschen der Zellen mit sterilem PBS 1x 3 mal. </li><li> 1 ml 10% Formalin, die Zellen zu fixieren. Legen Sie die Platte in Raumtemperatur für etwa 30 min. </li><li> Waschen der Zellen mit sterilem PBS 1x 3 mal. </li><li> Beflecken CD31, 1 ml steriler 1xPBS, das 10 &amp; mgr; l von CD31-FITC-Antikörper enthält. Um Fleck entweder VEGFR2, eNOS, VE-Cadherin oder Calponin, 1 mL sterile 1x PBS, die 4 ul VEGFR2 enthält, eNOS, VE-Cadherin oder Calponin primären Antikörper sind. </li><li> Inkubieren der Zellen auf Eis für 1 h in der Dunkelheit. </li><li> Entfernen der Antikörper, die durch die Zellen mit steriler PBS 3 mal waschen. </li><li> Für die Färbung von VEGFR2, eNOS, VE-Cadherin oder Calponin, 1 ml steriler 1x PBS, das 10 &amp; mgr; l FITC-konjugiertem anti-Kaninchen-IgG enthält. Inkubieren der Zellen auf Eis für 1 h in der Dunkelheit. Entfernen Sie die IgG, indem die Zellen mit steriler PBS 3 mal waschen. </li><li> Hinzufügen Hoechst 33342 bis zu einer Endkonzentration von 1 ug / ml, 10 min Inkubieren im Dunkeln. </li><li> Sehen Sie sich die Fluoreszenz von CD31, VEGFR2, eNOS, VE-Cadherin, Calponin (grün, Anregungswellenlänge von 518 bis 535 nm) und Hoechst (blau, Anregungswellenlänge von 361 nm) mit einem invertierten Fluoreszenzmikroskop. </li></ol></li></ol>

<ol>
	<li>Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+early+structural-functional+changes+in+the+endothelium+for+vascular+disease.">Implications of early structural-functional changes in the endothelium for vascular disease.</a> Arteriosclerosis, thrombosis, and vascular biology. 27, 266-274 (2007).</li><li>Cid, M. C., Segarra, M., Garcia-Martinez, A., Hernandez-Rodriguez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells,+antineutrophil+cytoplasmic+antibodies,+and+cytokines+in+the+pathogenesis+of+systemic+vasculitis.">Endothelial cells, antineutrophil cytoplasmic antibodies, and cytokines in the pathogenesis of systemic vasculitis.</a> Current rheumatology reports. 6, 184-194 (2004).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Reactive+protein-induced+endothelial+microparticle+generation+in+HUVECs+is+related+to+BH4-dependent+NO+formation.">C-Reactive protein-induced endothelial microparticle generation in HUVECs is related to BH4-dependent NO formation.</a> Journal of vascular research. 44, 241-248 (2007).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+circulating+CD31+/CD42-+microparticles+are+associated+with+impaired+systemic+artery+elasticity+in+healthy+subjects.">Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects.</a> American journal of hypertension. 20, 957-964 (2007).</li><li>Mackay, L. S., et al. <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+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema.">Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema.</a> Respiratory research. 14, 23 (2013).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> Journal of immunological methods. 244, 205-215 (2000).</li><li>Bowden, R. A., et al. <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+alpha4+integrin+and+VCAM-1+in+CD18-independent+neutrophil+migration+across+mouse+cardiac+endothelium.">Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.</a> Circulation research. 90, 562-569 (2002).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+induces+apoptosis+in+mouse+aortic+endothelial+cells+and+endothelial+dysfunction+in+mice+fed+high-calorie+and+high-cholesterol+diets.">Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets.</a> Life sciences. 92, 1165-1173 (2013).</li><li>Atkins, G. B., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+differentiation:+molecular+mechanisms+of+specification+and+heterogeneity.">Endothelial differentiation: molecular mechanisms of specification and heterogeneity.</a> Arteriosclerosis, thrombosis, and vascular biology. 31, 1476-1484 (2011).</li><li>Aitsebaomo, J., Portbury, A. L., Schisler, J. C., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brothers+and+sisters:+molecular+insights+into+arterial-venous+heterogeneity.">Brothers and sisters: molecular insights into arterial-venous heterogeneity.</a> Circulation research. 108, 929-939 (2008).</li><li>Shimamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+and+foods+on+contraction+of+endothelial+cells+as+a+key+mechanism+in+atherogenesis+and+treatment+of+atherosclerosis+with+endothelial-cell+relaxants+(cyclic+AMP+phosphodiesterase+inhibitors).">Drugs and foods on contraction of endothelial cells as a key mechanism in atherogenesis and treatment of atherosclerosis with endothelial-cell relaxants (cyclic AMP phosphodiesterase inhibitors).</a> Advances in experimental medicine and biology. 60, 77-105 (1975).</li><li>Chiu, J. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+disturbed+flow+on+vascular+endothelium:+pathophysiological+basis+and+clinical+perspectives.">Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives.</a> Physiological reviews. 91, 327-387 (2011).</li><li>Gimbrone, M. A., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction,+hemodynamic+forces,+and+atherogenesis.">Endothelial dysfunction, hemodynamic forces, and atherogenesis.</a> Annals of the New York Academy of Sciences. 902, 239-240 (2000).</li><li>Rostgaard, J., Qvortrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+demonstrations+of+filamentous+molecular+sieve+plugs+in+capillary+fenestrae.">Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.</a> Microvascular research. 53, 1-13 (1997).</li><li>Brutsaert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial-myocardial+signaling:+its+role+in+cardiac+growth,+contractile+performance,+and+rhythmicity.">Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity.</a> Physiological reviews. 83, 59-115 (2003).</li></ol>

The authors have nothing to disclose.

Diese Studie zeigt, ein einfaches Verfahren zur Isolierung und Kultur Endothelzellen von einer Maus Aorta ohne spezielle Ausrüstung. Die Immunfluoreszenzfärbung zeigte, daß die Mehrzahl der Zellen Endothelzellen nach dem zweiten Durchgang waren. Es wird vorgeschlagen , dass die Zellen am zweiten in den dritten Durchgang geeignet sind , in vitro und in vivo Experimenten endothelial biology zu studieren. Die EVP - ED aus dem Protokoll ...

Das vaskuläre Endothel nicht nur eine Barriereschicht ist , die Blut und Gewebe trennt, wird eine große endokrine Drüse betrachtet , die mit einer Oberfläche von 400 Quadratmetern 1 über den gesamten Gefäßbaum erstreckt. Das Wohlbefinden des Endothels ist wesentlich für vaskuläre Homöostase. Die dysfunktionalen Endothels beteiligt sich an verschiedenen kardiovaskulären Erkrankungen wie Atherosklerose, Vaskulitis und Ischämie / Reperfusion Verletzungen, usw. 2-4. Bis heute sind die spezifischen zellulären und molekularen Mechanismen in dieser Krankheitseinstellungen beteiligt sind nicht gut verstanden durch die diffuses anatomischen Natur des Endothels. Die Maus ist ein wichtiges Modell für die Forschung, da genetische Manipulationstechniken mehr bei Mäusen entwickelt als in jeder anderen Säugetierspezies. Jedoch ist die Isolierung von primären Maus-Aorta-Endothelzellen als besonders schwierig, da die geringe Größe der Aorta enzymat machtic Verdauung von Endothel nicht praktikabel. Einige Verfahren berichtet , zu isolieren und zu reinigen ECs 5-7 erfordern. Das Ziel dieses Protokolls ist es, ein einfaches Verfahren zu verwenden, um Endothelzellen aus der Maus Aorta zu isolieren und zu erweitern, ohne eine spezielle Ausrüstung. In diesem Protokoll wird die frisch isolierten Aorta in kleine Segmente geschnitten und ausgesät auf einer Matrix mit dem Endothel für endothelial Austrieb zu ermöglichen nach unten zeigt. Nach Segmenten entfernt werden, werden Endothelzellen in Endothel-bevorzugtes Medium erweitert und sind für Experimente nach zwei oder drei Passagen bereit. Die Vorteile des beschriebenen Verfahrens sind, dass: 1) beträchtlich hohe Anzahl von Endothelzellen aus einer einzigen Aorta geerntet werden; 2) die Lebensfähigkeit der Zellen ist gut erhalten; und 3) keine spezielle Ausrüstung oder Technik erforderlich ist. Sie stellt ein wirksames Mittel zur spezifischen zellulären und molekularen Mechanismen in Endothelzellen Pathophysiologie identifizieren. Für diejenigen, die bei der Untersuchung pr interessiert sindimary kultivierten Endothelzellen aus entweder Gen-Knock-out-Mäuse, Gen-Knock-in-Mäusen, oder einem murinen Krankheitsmodell ist dieses Protokoll sehr nützlich und leicht zu praktizieren.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

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.

Endothelzell Sprießen Spontane Endothelzellen aus einem Maus-Aorta-Segment gestartet sprießen. Die Maus-Aorta-Segment wurde für 4 Tage auf einem Wachstumsfaktor-reduzierte Matrix dann in endothelialen Zellwachstumsmedium wachsen gelassen. Die Endothelzellen Austrieb erscheint in der Regel innerhalb von 2 - 4 Tage. Photomikrographien wurden am Tag 4 (Figur 1) aufgenommen. Wie in den Abbildungen gezeigt, wandern zahlreiche Endothelzellen aus dem Segment ent...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

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

isolation-and-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.4K Aufrufe.  Wayne State University. 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.

Serie: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

Isolierung von primären murinen Gehirn mikrovaskulären Endothelzellen

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Forschung

Neurowissenschaften

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.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

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

Biologie

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolierung und Kultur Expansion von tumorspezifischen Endothelzellen

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Medizin

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;.

Isolierung der primären murinen Skelettmuskulatur mikrovaskuläre Endothelzellen

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

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.

Isolierung von embryonalen ventrikuläre Endothelzellen

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

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.

Isolierung von primären Lungenendothelzellen der Maus

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

Immunologie und Infektion

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.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

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 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.

Isolierung von Maus-Koronar-Endothelzellen

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 Aortic Endothelial Cells from Miniature Pigs

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

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

Isolation und Kultur der primären Aorten-Endothelzellen von Miniaturschweinen

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

Isolation and 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.

Isolierung und Charakterisierung von primären Leber-Sinus-Endothelzellen der Maus

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