Name: development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production
Uploaded: 2016-05-19T15:00:00
Description: Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

이 작품은 국립 심장, 폐, 혈액 연구소에 의해 지원되었다 HL56237, 당뇨병과 소화기의 국립 연구소 및 신장 질환 연구소 DK97391-03, 국립 과학 재단 (NSF-1227359 및 EPS-1003907)를 부여합니다.

1. 미세 유체 소자의 제작 <ol><li> SU-8 (50) 마스터 금형의 표준 포토 리소그래피 제작 <ol><li> 스핀 코팅 전에 실리콘 웨이퍼를 청소합니다. 15 분 동안 이소 프로필 알콜 (IPA) 다음에 15 분 동안 아세톤 노출 된 2 인치 실리콘 웨이퍼를 헹군다. 1 시간 동안 150 ℃에서 핫 플레이트 상에 배치하여, 웨이퍼 탈수. 탈수 후, 실온에서 웨이퍼를 냉각. </li><li> 스핀 코트 SU-8 포토 레지스트와 실리콘 웨이퍼. 웨이...

<ol>
	<li>Curry, F. R. E., Adamson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+modulation+at+the+cell,+microvessel,+or+whole+organ+level:+towards+closing+gaps+in+our+knowledge.">Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge.</a> Cardiovasc Res. 87, 218-229 (2010).</li><li>Michel, C. C., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+permeability.">Microvascular permeability.</a> Physiol Rev. 79, 703-761 (1999).</li><li>Rogers, J. A., Nuzzo, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+soft+lithography.">Recent progress in soft lithography.</a> Mater Today. 8, 50-56 (2005).</li><li>Sackmann, E. K., Fulton, A. L., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+present+and+future+role+of+microfluidics+in+biomedical+research.">The present and future role of microfluidics in biomedical research.</a> Nature. 507, 181-189 (2014).</li><li>Li, X., Xu, S., He, P., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+recapitulation+of+functional+microvessels+for+the+study+of+endothelial+shear+response,+nitric+oxide+and+[Ca2+]I.">In vitro recapitulation of functional microvessels for the study of endothelial shear response, nitric oxide and [Ca2+]I.</a> PLoS One. 10, 0126797 (2015).</li><li>He, P., Pagakis, S. N., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytoplasmic+calcium+in+single+microvessels+with+increased+permeability.">Measurement of cytoplasmic calcium in single microvessels with increased permeability.</a> Am J Physiol. 258, 1366-1374 (1990).</li><li>Zhou, X., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+measurements+of+intracellular+nitric+oxide+in+intact+microvessels+using+4,5-diaminofluorescein+diacetate.">Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate.</a> Am J Physiol-Heart C. 301, 108-114 (2011).</li><li>Yuan, D., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+remodeling+alters+adhesion+protein+and+cytoskeleton+reactions+to+inflammatory+stimuli+resulting+in+enhanced+permeability+increases+in+rat+venules.">Vascular remodeling alters adhesion protein and cytoskeleton reactions to inflammatory stimuli resulting in enhanced permeability increases in rat venules.</a> Journal of Applied Physiology. 113, 1110-1120 (2012).</li><li>He, P., Zhang, X., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++entry+through+conductive+pathway+modulates+receptor-mediated+increase+in+microvessel+permeability.">Ca2+ entry through conductive pathway modulates receptor-mediated increase in microvessel permeability.</a> Am J Physiol. 271, 2377-2387 (1996).</li><li>Zhou, X., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+[Ca2+]i+and+caveolin-1+antagonistically+regulate+eNOS+activity+and+microvessel+permeability+in+rat+venules.">Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules.</a> Cardiovasc Res. 87, 340-347 (2010).</li><li>Zhu, L., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-activating+factor+increases+endothelial+[Ca2+]+i+and+NO+production+in+individually+perfused+intact+microvessels.">Platelet-activating factor increases endothelial [Ca2+] i and NO production in individually perfused intact microvessels.</a> Am J Physiol-Heart C. 288, 2869-2877 (2005).</li><li>He, P., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization+modulates+endothelial+cell+calcium+influx+and+microvessel+permeability.">Depolarization modulates endothelial cell calcium influx and microvessel permeability.</a> Am J Physiol. 261, 1246-1254 (1991).</li><li>He, P., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+hyperpolarization+increases+[Ca2+]i+and+venular+microvessel+permeability.">Endothelial cell hyperpolarization increases [Ca2+]i and venular microvessel permeability.</a> J Appl Physiol. 76 (1985), 2288-2297 (1994).</li><li>Xu, S., Zhou, X., Yuan, D., Xu, Y., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolin-1+scaffolding+domain+promotes+leukocyte+adhesion+by+reduced+basal+endothelial+nitric+oxide-mediated+ICAM-1+phosphorylation+in+rat+mesenteric+venules.">Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules.</a> Am J Physiol Heart Circ Physiol. 305, 1484-1493 (2013).</li><li>Zadeh, M. H., Glass, C. A., Magnussen, A., Hancox, J. C., Bates, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-Mediated+Elevated+Intracellular+Calcium+and+Angiogenesis+in+Human+Microvascular+Endothelial+Cells+In+Vitro+are+Inhibited+by+Dominant+Negative+TRPC6.">VEGF-Mediated Elevated Intracellular Calcium and Angiogenesis in Human Microvascular Endothelial Cells In Vitro are Inhibited by Dominant Negative TRPC6.</a> Microcirculation. 15, 605-614 (2008).</li><li>Tsai, 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=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> The Journal of clinical investigation. 122, 408-418 (2012).</li><li>Kim, S., Lee, H., Chung, M., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+functional,+perfusable+3D+microvascular+networks+on+a+chip.">Engineering of functional, perfusable 3D microvascular networks on a chip.</a> Lab Chip. 13, 1489-1500 (2013).</li><li>D'Amico Oblak, T., Root, P., Spence, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+monitoring+of+ATP-stimulated,+endothelium-derived+nitric+oxide+production+in+channels+of+a+poly(dimethylsiloxane)-based+microfluidic+device.">Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device.</a> Analytical chemistry. 78, 3193-3197 (2006).</li><li>Zhou, X. P., Yuan, D., Wang, M. X., He, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2O2-induced+endothelial+NO+production+contributes+to+vascular+cell+apoptosis+and+increased+permeability+in+rat+venules.">H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules.</a> Am J Physiol-Heart C. 304, 82-93 (2013).</li><li>Lee, P. J., Hung, P. J., Rao, V. M., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter+scale+microbioreactor+array+for+quantitative+cell+biology.">Nanoliter scale microbioreactor array for quantitative cell biology.</a> Biotechnol Bioeng. 94, 5-14 (2006).</li><li>Zakrzewicz, A., Secomb, T. W., Pries, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angioadaptation:+Keeping+the+Vascular+System+in+Shape.">Angioadaptation: Keeping the Vascular System in Shape.</a> Physiology. 17, 197-201 (2002).</li><li>Lipowsky, H. H., Kovalcheck, S., Zweifach, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+blood+rheological+parameters+in+the+microvasculature+of+cat+mesentery.">The distribution of blood rheological parameters in the microvasculature of cat mesentery.</a> Circulation Research. 43, 738-749 (1978).</li><li>Pisano, M., Triacca, V., Barbee, K. A., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+model+of+the+tumor-lymphatic+microenvironment+with+simultaneous+transendothelial+and+luminal+flows+reveals+mechanisms+of+flow+enhanced+invasion.">An in vitro model of the tumor-lymphatic microenvironment with simultaneous transendothelial and luminal flows reveals mechanisms of flow enhanced invasion.</a> Integr Biol (Camb). 7, 525-533 (2015).</li><li>McCann, J. A., Peterson, S. D., Plesniak, M. W., Webster, T. J., Haberstroh, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-uniform+flow+behavior+in+a+parallel+plate+flow+chamber+:+alters+endothelial+cell+responses.">Non-uniform flow behavior in a parallel plate flow chamber : alters endothelial cell responses.</a> Ann Biomed Eng. 33, 328-336 (2005).</li><li>Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+venular+microvessel+permeability+by+calcium+influx+into+endothelial+cells.">Modulation of venular microvessel permeability by calcium influx into endothelial cells.</a> FASEB J. 6, 2456-2466 (1992).</li><li>Kojima, H., 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=Detection+and+imaging+of+nitric+oxide+with+novel+fluorescent+indicators:+diaminofluoresceins.">Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins.</a> Anal Chem. 70, 2446-2453 (1998).</li><li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442, 368-373 (2006).</li><li>Lamberti, G., 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=Bioinspired+microfluidic+assay+for+in+vitro+modeling+of+leukocyte-endothelium+interactions.">Bioinspired microfluidic assay for in vitro modeling of leukocyte-endothelium interactions.</a> Anal Chem. 86, 8344-8351 (2014).</li><li>Myers, D. R., 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=Endothelialized+microfluidics+for+studying+microvascular+interactions+in+hematologic+diseases.">Endothelialized microfluidics for studying microvascular interactions in hematologic diseases.</a> J Vis Exp. , (2012).</li><li>Smith, A. M., Prabhakarpandian, B., Pant, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Shear+Adhesion+Map+Using+SynVivo+Synthetic+Microvascular+Networks.">Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks.</a> Jove-J Vis Exp. , e51025 (2014).</li><li>Booth, R., Kim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+microfluidic+in+vitro+model+of+the+blood-brain+barrier+(iBBB).">Characterization of a microfluidic in vitro model of the blood-brain barrier (iBBB).</a> Lab on a Chip. 12, 5282-5282 (2012).</li><li>Shao, J. B., 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=Integrated+microfluidic+chip+for+endothelial+cells+culture+and+analysis+exposed+to+a+pulsatile+and+oscillatory+shear+stress.">Integrated microfluidic chip for endothelial cells culture and analysis exposed to a pulsatile and oscillatory shear stress.</a> Lab on a Chip. 9, 3118-3125 (2009).</li><li>Yeon, J. H., 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=Reliable+permeability+assay+system+in+a+microfluidic+device+mimicking+cerebral+vasculatures.">Reliable permeability assay system in a microfluidic device mimicking cerebral vasculatures.</a> Biomed Microdevices. 14, 1141-1148 (2012).</li><li>Golden, A. P., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microfluidic+hydrogels+using+molded+gelatin+as+a+sacrificial+element.">Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element.</a> Lab Chip. 7, 720-725 (2007).</li><li>Zheng, 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=In+vitro+microvessels+for+the+study+of+angiogenesis+and+thrombosis.">In vitro microvessels for the study of angiogenesis and thrombosis.</a> Proc Natl Acad Sci U S A. 109, 9342-9347 (2012).</li><li>Baker, B. M., Trappmann, B., Stapleton, S. C., Toro, E., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+embedded+within+extracellular+matrix+to+define+vascular+architectures+and+pattern+diffusive+gradients.">Microfluidics embedded within extracellular matrix to define vascular architectures and pattern diffusive gradients.</a> Lab Chip. 13, 3246-3252 (2013).</li></ol>

<html> 이 글에서, 우리는 배양 된 미세 혈관 네트워크의 개발을위한 상세한 프로토콜을 제시, EC 접합 및 F-굴지의 유통 세포 골격 특성화 및 EC의 정량적 측정 [칼슘 2 +] i와 미세 유체 장치를 이용하여 NO 생산. 관류 미세 유체 장치는 생체 내 미세 혈관 형상과 전단 유동 조건의 가까운 시뮬레이션을 허용하는 체외 모델을 제공합니다. 배양 된 미세 혈관 네트워크가 일차...

생체 내에서 혈관 벽을 라이닝 내피 세포 (EC에)는 끊임없이 유동에 노출되어 있지만, 배양되는 EC 종종 정적 조건 하에서 성장 및 염증성 표현형 1,2 나타내고있다. 미세 유체 기술은 원하는 유량 조건 하에서 성장하기 위하여, 특히 혈관의 EC를 들어, 세포 배양을위한 기회를 제공하는 마이크로 기하학적 제약 (서브 밀리미터) 채널 (3)을 통해 유체를 정밀하게 제어 할 수있다. 이러한 기능은 기존의 정적 차원 세포 배양에 비해 생체 내에서의 세포 배양 조건을 가까이합니다. 그들은 마이크로 유체 장치 vasculatures의 다른 유형을 모델링하는 데 사용되는 기계적 및 / 또는 화학적 자극에 대한 EC의 반응을 연구 할 때 매우 중요하다. 정적 세포 배양, 생물 의학 F의 적응과 미세 유체의 응용 프로그램을 통해 마이크로 네트워크에 의해 입증 장점에도 불구하고의 ield 제한 남아있다. 최근의 검토에 의해보고되는 경우,이 필드 (85 %)의 서적의 대부분은 공학 저널 (4)에 정지한다. 미세 유체 장치의 성능은 대부분의 생물 학자들이 같은 트랜스 웰 분석이 소형 장치에 매크로 규모의 배양 접시 / 유리 슬라이드로 현재 기술로 전환하기에 충분한 납득되지 않았습니다. 미세 유체 앞으로이 필드를 이동하는 학제 간 협력을 필요로하는 종합 분야입니다. 이 기술 문서의 목적은 생물학적 응용 및 미세 미세 혈관의 기능 검증을 제공하면서, 기술 분야 사이의 갭을 ​​감소시키고 생물에 의한 제조 방법을 이해할 수 있도록하는 것이다. 시각화 실험 프로토콜은 마이크로 유체 장치 및 엔지니어와 생물 학자들 사이의 긴밀한 협력을 나타내는 생물학적 유틸리티, 모두의 제조를 포함한다. 우리는 최근 몇 가지를보고미세 유동 장치 (5) 시험 관내 미세 혈관 망을 이용하여 생물학적 응용. 적절히 원하는 전단 응력을 마이크로 네트워크의 치수를 설계 및 적용하기 위해서, 수치적인 모델 밀접 유량 프로파일을 추정하는 전산 유체 역학 소프트웨어를 사용하여 구축 하였다. 즉, 합류에 도달 마이크로에 접종 한 기본 인간 제대 정맥 내피 세포 (HUVECs를)은 연속 관류 3-4 일, 마이크로의 전체 내부 표면에 덮여있다. 적절한 배리어 형성 정적 세포 배양 조건에서 미세 혈관 손상에 형성된 것과 비교 VE-cadherin의 염색에 의해 증명되었다. 개별적으로 관류 그대로 미세 혈관 6-8에서 개발 된 실험 프로토콜을 적용하여, 우리는 정량적으로 EC의 변화를 측정 [칼슘 2 +] i와 산화 질소 (NO) 형광에서와 아데노신 삼인산 (ATP)에 응답하여 생산에 모아와 공 초점 및 기존의 형광 현미경. EC의 효능 유발 증가 [칼슘 2 +] i와 NO 생산은 미세 혈관 투과성 6-15에 염증 매개체에 의한 증가에 필요한 세포 내 신호로보고되고있다. 일부 이전 연구는 DAF-2 DA로드 미세 유체 장치 (16, 17)의 이미지를 보여 주었다 있지만, 적절한 해상도와 데이터 분석은 아직 18을 달성하지 않았다. 우리의 지식으로,이 연구는 내피 세포에 작용 물질에 의한 동적 변화 [칼슘 2 +] i와 NO 생산 이용한 미세 유체 기반 시스템의 첫 번째 양적 측정을 보여줍니다. 미세 가공 기술이 몇 마이크론 마이크로 채널을 제조 및 생체 내 미세 혈관의 형상을 모방하는 복잡한 패턴의 개발을 가능하게 할 수있는 유연성을 가지고있다. 여기에서 우리는 분기의 세 가지 수준으로 일반적인 마이크로 채널 네트워크를 발표했다. 이네트워크는 미세 크린룸 소프트 리소그래피에서 수행되는 리소그래피의 조합에 의해 제조된다.

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">ATP</td>
			<td style="width:247px;">Sigma-Aldrich</td>
			<td style="width:327px;">A2383</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Fisher Scientific</td>
			<td>A929</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biopsy punch</td>
			<td>Miltex</td>
			<td>33-31 AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Albumin</td>
			<td>MP Biomedicals</td>
			<td>810014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Brain Extract (BBE)</td>
			<td>Lonza</td>
			<td>CC-4098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover-slip</td>
			<td>Fisher Scientific</td>
			<td>12-542C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAF-2 DA</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-Goat IgG (H+L) Secondary Antibody</td>
			<td>Life technologies</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DRAQ5 (nuclei staining)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>4084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td>Sigma-Aldrich</td>
			<td>E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluo-4 AM</td>
			<td>Life technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750-078</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-548B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Pasteur pippette</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-10KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human umbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl alcohol (IPA)</td>
			<td>VWR</td>
			<td>89125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mammalian Ringer Solution Ingredient</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl (132 mM)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL (4.6 mM)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 &#183; 2H2O (2.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4 &#183;7H2O (1.2 mM)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose(5.5 mM)</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3 (5.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Salt (9.1 mM)</td>
			<td>Research Organics</td>
			<td>6007H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Acid (10.9 mM)</td>
			<td>Research Organics</td>
			<td>6003H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB 131 Culture Medium</td>
			<td>Life technologies</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin (F-actin staining)</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline&#160;</td>
			<td>Life technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>Exel Int</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scotch tape</td>
			<td>3M</td>
			<td>34-8711-3070-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicon wafer</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2050 Y111072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tissue culture flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z707503-100EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Chemical Book</td>
			<td>T6878</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin Neutralizer solution</td>
			<td>Gibco</td>
			<td>R-002-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin/EDTA Solution (TE)</td>
			<td>Gibco</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Cole-Parmer</td>
			<td>PTFE microbore tubing, 0.012&#34; ID x 0.030&#34; OD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VE-cadherin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Laminar hood</td>
			<td>NuAire</td>
			<td>NU-425 Class II, Type A2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD camera</td>
			<td>Hamamatsu</td>
			<td>ORCA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art</td>
			<td>F42022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>Wenesco</td>
			<td>HP-1212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp oven</td>
			<td>Barnstead</td>
			<td>3608-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithography bench</td>
			<td>Karl Suss</td>
			<td>MA6 Contact Lithography</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical microscope</td>
			<td>Nikon</td>
			<td>L200 ND &#38; Diaphod 300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shutter for the CCD camera</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>PVA TePla/Harrick plasma</td>
			<td>M4L/PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Brewer Science</td>
			<td>Cee 200X</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump system</td>
			<td>Harvard Apparatus</td>
			<td>703005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Software</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD 2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFD simulation software</td>
			<td>COMSOL</td>
			<td>COMSOL Multiphysics 4.0.0.982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Images acquire and analyse for NO production&#160;</td>
			<td>Universal Imaging</td>
			<td>Metafluor</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production

생체 내에서 혈관 벽을 라이닝 내피 세포 (EC에)는 끊임없이 유동에 노출되어 있지만, 배양되는 EC 종종 정적 조건 하에서 성장 및 염증성 표현형을 나타내고있다. 미세 유체 장치의 개발이 20 년간 엔지니어들이 채택되었지만, 생물학적 응용은 제한 남아있다. 생물학적 응용 프로그램에 의해 검증보다 생리 학적으로 관련 시험 관내 미세 혈관 모델들이 필드에 발전 생체 사이 시험관 연구에서 격차를 해소하는 것이 중요하다. 여기서는 장기 관류 용량을 갖는 미세 유동 장치를 이용하여 배양 된 미세 혈관 네트워크의 개발을위한 상세한 절차를 제시한다. 또한, 종래의 공 초점 및 형광 현미경을 사용하여 실시간으로 EC [칼슘] i 및 산화 질소 (NO)의 생산에서 작용제 - 유도 변화의 정량적 측정을위한 응용 프로그램을 보여준다. 형성된 미세 혈관 순연속 관류와 작업이되는 EC 사이의 잘 발달 된 접합을 보여 주었다. 분포를-cadherin의 VE 것은 정적 배양 EC 단층보다 그대로 미세 혈관에서 관찰에 가까운이었다. EC의 일시적인 증가가 유도 ATP [CA는 2+] i 및 NO 생산은 정량적 배양 된 미세 혈관의 기능을 검증 개별 셀 레벨에서 측정되었다. 이 마이크로 유체 장치는 내장 컴퓨터가 기존의 정적 2D 문화보다 생체 내에서의 세포 배양 환경을 가까이하게 잘 조절, 생리 학적으로 관련 흐름에 따라 증가 할 수 있습니다. 마이크로 채널 네트워크 설계 다용도이며, 제조 공정이 단순하고 반복적이다. 이 장치는 쉽게 고해상도 영상화를 가능하게하는 공 초점 현미경 또는 종래의 시스템에 통합 될 수있다. 배양 된 미세 혈관 네트워크가 일차 인간 EC에 의해 형성 될 수 있기 때문에, 가장 중요한 것은,이 방법은 방법을 조사하기 위해 유용한 도구가 될 것병리학 적으로 환자 샘플에서 변경된 혈액 성분은 인간의 내장 컴퓨터에 영향을 미치는 임상 문제에 대한 통찰력을 제공합니다. 또한, 약물 스크리닝을위한 플랫폼으로 개발 될 수있다.

이 섹션에서는이 프로토콜을 개발 한 배양 된 미세 혈관 네트워크와 얻어진 결과의 일부를 보여줍니다. 마이크로 패턴은 세 개의 레벨 분기 네트워크 (도 1a)이다. 이 설계에서, 네 개의 100 ㎛ 폭 보조 채널로 다시이 126 μm의 넓은 채널, 나뭇 가지로 159 ㎛ 폭 어머니 채널 분기합니다. 의 3D 수치 시뮬레이션이 마이크로 네트워크 내에 전단 응력의 ​​세 가지 레...

Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

개발 및 특성화의<em&gt; 체외</em&gt; 미세 혈관 네트워크 및 내피의 정량적 측정 [칼슘<sup&gt; 2 +</sup&gt;]<sub&gt; 전</sub&gt; 및 질소 산화물 생산

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

The overall goal of this procedure is to develop a microfluidic device that allows endothelial cells to grow under physiologically relevant shear flow and to measure the agonist induced changes in their calcium and and nitric oxide production. This message can help advance the future of microvessel research, by validating the in vitro microvessel model and bridging the gaps between in vivo and the in vitro studies. The main advantages of this technique are the versatile design of the microchannels to mimic various vasculature and they improve cell culture environment that is closer to in vivo than the conditional static to that original cultures.Demonstrating the procedures will be Sulei and Xiang, the two from my laboratory. Before beginning, use standard photolithography to create a master mold and soft lithography to fabricate the PDMS microchannel devices as described in the text protocol. To prepare the microchannel devices, first cover the inlet and outlet with a thin piece of PDMS and place the device inside a Petri dish with a wet tissue.Then, wrap the dish with Parafilm and sterilize the device under a UV light for at least three hours. The next step is to apply the fibronectin. First, place a drop of 30 to 50 microliters of PBS at the inlet.Create a vacuum at the outlet to rinse the device. Next, place a drop of 100 micrograms per milliliter fibronectin at the inlet. Create a vacuum at the outlet to load the fibronectin solution.Next, cover the inlet and outlet. Wrap the dish with Parafilm again and incubate the device overnight at four degrees Celsius. The next day, manually rinse the device with PBS three times.Then, load it with 30 to 50 microliters of cell culture media and warm up the device in an incubator at 37 degrees Celsius for at least 15 minutes. Begin by mixing HUVECs into HUVEC culture media with eight percent Dextran at two to four million cells per milliliter. The Dextran is needed to increase the viscosity and thus improve the cell seeding.Next, put 10 to 20 microliters of cells over the inlet and introduce them using gravity or by using capillary action from a glass pasteur pipette at the outlet. Next, incubate the device for 15 to 20 minutes. Then, check the seeding status under a microscope.If necessary, load more cells to achieve a desired cell density. Once enough cells are loaded, gently rinse the device with normal, warm cell culture medium to remove the Dextran. Then, culture the device without any media perfusion for six hours.Next, set up the tubing connections for the long term perfusion. Tape the device to a Petri dish and then connect the inlet tubing to a syringe with a needle. Insert tubing into both the inlet and the outlet of the device.Connect the outlet tubing to a waste collector. Now, place the dish and waste container in an incubator. Connect the syringe with a long inlet tube to an external perfusion pump that passes through the incubator door.Then, set the perfusion rate according to the experimental design. With a well controlled perfusion, the culture in the microvessel network can be maintained up to two weeks. Therefore, it can be extended to longer term studies mimicking the cellular and the hemodynamic environment on the different physiological and the pathological condition.To begin the immuno-staining, such as VE-cadherin labeling, first fix the cells by perfusing with two percent paraformaldehyde for 30 minutes at four degrees Celsius. After a series of perfusions to block, permeabilize, and stain with antibodies, keep the device at four degrees Celsius until the cells are imaged. To image the calcium concentration in the cells, perfuse the device with 10 milligrams per milliliter of albumin in Ringer solution for 15 minutes at 37 degrees Celsius.Next, perfuse the device with five micromolar Fluo-4 AM for 40 minutes at 37 degrees Celsius. Then, wash out the lumen bound Fluo-4 AM with albumin Ringers for 15 minutes at 37 degrees Celsius. Next, start up the confocal system for intercellular calcium level imaging with Fluo-4 AM.Acquire images of the Fluo-4 loaded microvessels.Collect the baseline images for 10 minutes. Then, perfuse the device continuously with 10 micromolar ATP and record the changes of fluorescence intensity for 20 minutes. To image nitric oxide production in the cells, perfuse the device with albumin Ringers for 15 minutes at 37 degrees Celsius.Then, perfuse the cells with 5 micromolar DAF-2 DA for about 35 to 40 minutes at 37 degrees Celsius. Next, set up the fluorescence imaging system to measure ATP induced nitric oxide production. Record the baseline for 5 minutes, then perfuse the device with 10 micromolar ATP and collect the images for 30 minutes at one minute intervals.For the cell nitric oxide measurement, it is important to understand that the detach adapter for intensity profile represents a cumulative nitric oxide production with time and not a nitric oxide concentration. Manually select the region of interests from the collected images at the individual cell level. Each ROI covers the area of one individual's cell which can be indicated by the floresense outline.Quantify the ATP induced changes in endothelial calcium and nitric oxide. By calculating the changes in the floresense intensity of Fluo-4, less the tissue background. Quantify the nitrous oxide production rate by conducting the first differential conversion of the floresense intensity of DAF-2 over time.The microchannel pattern in the device has a three level branching network. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 microliters per minute. Endothelial cells were seeding into the network as described.Then, F-actin and nuclei were labeled. Similarly, VE-cadherin was also stained. The results were similar to the VE-cadherin expression in an intact venule.Next, ATP induced increases in intracellular calcium and nitric oxide production were examined. Calcium levels were examined using Fluo-4 AM as described in the protocol section. Nitric oxide production was monitored using DAF-2 DA as described in the protocol section.The results were comparable to those derived with individually perfused intact venules. The advanced was easy and tightly controlled in biological conditions and the dynamic fluid environments, which would not have been possible with conventional microskill techniques. After watching this video, you should have a good understanding of how to fabricate the device, and perform calcium and nitric oxide measurements.After this technique paved the way for researchers, not only to investigate underlying agonist induced but also to open broader applications for bio medical research.

Research

JoVE Journal

Biology

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

질소 산화물과 세포로부터 전자 상자 성체의 프로그램 공명 분광학에 의한 초과 산화물 음이온 급진적가 탐지 스핀 트랩을 사용하여

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

연구

생물학

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

라이신 Demethylase 활동의 양적 측정을위한 MassSQUIRM의 응용

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Analysis of Oxidative Stress in Zebrafish Embryos

제브라 피쉬 배아에서 산화 스트레스의 분석

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

세포 내 칼슘의 두 광자 이미징<sup&gt; 2 +</sup&gt; 고립 된 쥐 대동맥의 취급 및 내피 세포에서 산화 질소 생산과 부드러운 근육 세포

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

본래 동맥의 내피 층에서 산화 질소와 활성 산소의 욕실 얼굴 인식

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

화학 발광 법을 사용하여 생체 물질에서 산화 질소 통로에서 아질산염과 질산염, 대사 산물을 측정

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

유전자 인코딩 된 형광 산화 질소의 응용 프로그램 (• NO) 단일 세포에서 NO • 신호의 실시간 이미징을위한 프로브의 geNOps,

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

정량적 이미징 기법을 이용한 이종 세포 집단의 차별화 및 특성 규명

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...