이 작품은 마취와 수술 전후 관리의 UCSF 부에 의해 지원되었다.

1. 도금 및 미세 혈관 내피 세포의 유지 보수 <ol><li> 해동 및 제조 업체에 따라 혈관 내피 세포 성장 지침을 제공. 이 프로토콜에서는, 세포는 EGM-2 MV 미디어 (론자)에서 재배되었고, 그것은 모든 실험은 통과 번호로 인한 변형을 최소화하기 위해 해동 후 2 주 내에서 수행하는 것이 좋습니다. HMVEC 폐를 가진 우리의 실험은 9-4 구절 사이에 수행됩니다. </li><li> 1 일 : 세포가 80 % ~ 90 % confluency에 도달하면 trypsinize 및 ML 당 120,000 세포에 resuspend을. 48도, 폴리스티렌 조직 배양 플레이트 (들)에 플레이트 잘 당 36,000 세포 (0.3 mL를). 배경 형광 판독 값을 얻기 위해 백혈구와 함께 배양되지 않습니다 HMVEC의 우물이 (가) 있습니다. 특히 형광 분석, 교대 행 또는 열을 위해 디자인 된 48 - 웰 플레이트를 사용하지 않는 이웃 우물에서 어떤 빛 ​​오염을 최소화 할 수 있습니다. 참고 : 웰스 폴리 라이시로 사전 코팅 할 수 있습니다NE, 콜라겐이나 fibronectin의. </li><li> 주 2 : 신선한 매체를 추가합니다. </li><li> 3 일 : 위상차 현미경으로 세포가 (즉, &quot;조약돌&quot;외관과 작은 세포 파편) 건강한 확인하고, 그들은 100 % 합류 (그림 1)입니다. 세포가 100 % 합류하지 않은 경우 그들은 준비가 될 때까지 매일 미디어를 변경합니다. </li><li> HMVEC 단층 치료 준비가되면, 행크의 균형 소금 솔루션 (HBSS) 한 번 세포를 세척하고 적절한 우물에 염증 작용제를 포함하는 신선한 EGM-2 MV 미디어 또는 미디어 0.3 ML를 추가합니다. 이 프로토콜에 HMVEC 폐는 TNFα 처리 하였다 3 시간 동안 [100 NG / ML] (PeproTech, 뉴저지)이 EC에 21 잘 설명 활성제이기 때문에. 주 : 당신의 활성화 HMVEC 세포 (단계 4.1)와 calcein 레이블이 호중구 (단계 3.5) 오전 그래서 당신은 시간 실험을하는 것은 동시에 사용할 준비가 권장됩니다. 그것은 neutroph를 분리하고 라벨을 우리에게 약 2.5 시간 소요ILS. </li></ol> 2. Polymorphprep를 사용하여 호중구의 분리 <ol><li> 이전 22 설명 된 호중구를 분리, 또는 전체 혈액에서 다형 과립구를 분리하는 기성품 밀도 기울기 솔루션. Nuzzi, 외 의해 프로토콜에 따라 Polymorphprep을 채용. 전체 혈액의 23 ML 당 약 2-3 X 10 6 호중구의 생산량 2007 결과. 참고 : 적혈구를 제거하기 위해 과도한 적혈구 용해, 덱스 트란 침강을 방지하려면이 밀도 기울기 원심 분리 24 이전에 수행 할 수 있습니다. </li><li> RT에 Polymorphprep 밀도 기울기 솔루션을 가지고. </li><li> 의 면전에서 건강한 인간 자원에서 전체 혈액의 30 ML를 수집 헤파린, 구연산 또는 EDTA로 항 응고. 혈액은 2 시간 이내에 사용하고, 18-22 ° C. 사이에서 유지되어야한다 </li><li> 15 ML 원뿔 튜브의 밀도 기울기 용액 (5 ㎖를 통해 전체 혈액의 5 층 ML <strong&gt; 그림 2A). 이 다음 원심 분리 조건을 변경할 수 있으므로 혈액의 볼륨과 밀도 기울기 솔루션을 변경하지 마십시오. </li><li> 18-22시 30 분 450 XG에 원심 분리기 ° C. 천천히 속도를 아래로 원심 분리기 (즉, 원심 브레이크를 해제) 및 호중구의 활성화를 줄이고 레이어의 혼합을 방지하기 위해 진동을 방지하기 위해 잘 튜브의 균형을해야합니다. 이상 원심 분리 시간 이상 원심력 펠렛 적혈구쪽으로 더 아래로 마이그레이션하는 호중구를 포함하는 낮은 백혈구 층에서 발생합니다. </li><li> 호중구 (그림 2B)를 포함하는 레이어의 오염을 방지하기 위해 말초 혈액을 포함하는 플라즈마 상 백혈구 밴드를 대기음. </li><li> 호중구를 포함하는 낮은 백혈구 층을 제거하는 1-2 ML 혈청 피펫을 사용합니다. PBS의 30 ML에 호중구 층을 결합 50ml에 (칼슘없이 + 및 마그네슘 2 +)원뿔 튜브. </li><li> PBS 50 ML (칼슘없이 + 및 마그네슘 2 +) 18-22시 10 분 450 XG에 원심 분리기 ° C.에 볼륨을 가지고 </li></ol> 참고 : 단계 2.9 및 2.10은 선택 사항이지만 매우 좋습니다. <ol start="9"><li> 뜨는을 대기음. 적혈구를 용해하기 위해 30 초 동안 멸균 물 8 ㎖에 호중구를 resuspend을 즉시 배 PBS 2 ML (칼슘없이 + 및 마그네슘 2 +) 부드럽게 손으로 혼합이.에서 세포를 배양하지 마십시오에 세포 현탁액을 추가 이상 30 초 동안 물 상당한 호중구 죽음이 발생할 수 있기 때문이다. </li><li> 18-22에서 5 분 250 XG에 원심 분리기 ° C. </li><li> PBS의 10 ML (칼슘없이 + 및 마그네슘 2 +)과 18-22에서 5 분 250 XG에서 다시 원심 분리기 ° C. 한 번 세포를 씻으 </li><li> RPMI-1640 10 ㎖에 호중구를 resuspend을 (페놀 레드없이) 트리 판 블루를 사용하여 살아있는 세포를 계산E 염색 제외 방법. 이 호중구의 활성화가 발생할 수 있으므로 ML 당 5 × 10 6보다 큰 세포 밀도의 연장 기간을 피하십시오. </li></ol> 3. Calcein AM와 호중구 레이블 <ol><li> 6 × 10 5 호중구는 48 - 웰 플레이트 잘 당 사용됩니다. </li><li> 계산 후, 50 ML 원뿔 튜브에 재 부유 세포를 전송하고 2-4 페놀 레드 무료 RPMI-1640 ML 당 X 10 6 세포 호중구를 희석. </li><li> calcein 3 μM (calcein AM 품절 즉, 2.98 μL (1 MG / ML) 당 ML 미디어)의 주식 솔루션 AM 추가하고 부드럽게 튜브를 가볍게 치면 잘 섞는다. 이상 5 μM는 calcein 호중구에서 누수가 발생합니다 오전. 주 : 비 형광 calcein AM이 높은 형광 분자 calcein (즉, 죽은 세포가 형광을하지 않을 것이다) 생산 내생 에스 테라 제에 의해 세포 내에서 쪼개 있습니다. </li><li> 알루미늄 호일로 튜브를 커버와 f를 품다또는 37 30 분 ° 어둠 주에 C :. 긴 배양 시간은 접착 분석의 과정을 통해 호중구에서 해방되고 더 calcein이 발생할 수 있습니다. </li><li> 18-22 ° C에서 5 분 450 XG에 원심 분리기 및 RPMI-1640 10 ㎖ (페놀 레드 않고, 37 미리 예열 ° C)의 2 배 호중구을 씻는다. 40 μM 살균 필터가 18-22에서 덩어리를 제거하고 5 분 450 XG에 한 번 더 원심 분리 할 수​​ 있지만 두 번째 세척에 대한 세포를 현탁 한 후, 최초의 호중구를 통과 ° C. </li><li> ML 당 2 × 10 6 호중구에서. (37 ° C로 예열없이 페놀 레드) RPMI 1640 호중구를 resuspend을 </li></ol> 4. 호중구 바인딩 분석 <ol><li> 필터 소독 (0.22 μm의) RPMI 1640 0.5 ML (페놀 레드없이) 잘 당 3 % BSA를 포함하는와 배 HMVEC 단일 층을 씻으십시오. </li><li> 미디어를 대기음 6 × 10 5 calcein - 라벨 호중구 (셀 suspensio의 0.3 ML를 추가. 48 - 웰 플레이트주의의 적절한 우물에 호중구가없는 RPMI 1640 N), 0.3 ML은 (페놀 레드없이) : 이것은 cm 2 당 8 × 10 5 호중구에 해당합니다. </li><li> 37 ° C에서 20 분, 5 % CO 2 배양기에 대해 품어. </li><li> 총 호중구 형광 (PRE 세척) : 485 ㎚의 여기 파장과 FLUOstar, 또는 이와 동등한 분광 광도계 520 nm의 (형광 필터 세트)의 발광 파장 각 우물의 형광 강도를 측정한다. . 직전에 배양 엔드 포인트에 참고이 단계를 수행하십시오 :이 프로토콜에서는, calcein의 여기 및 방출되는 빛의 검출이 밝혀 우물의 상단에서 수행되었다, 그러나 모두는 우물의 바닥에서 수행 할 수 있습니다. </li><li> PBS의 잘 당 0.5 mL로 배 HMVEC 단층 및 호중구가 포함 된 우물 세척 (W / CA 2 + 및 마그네슘 2 +). 미디어 및 세척을 제거반전 플레이트 및 종이 타월로 가볍게 두드려하면 남아있는 액체를 제거합니다. </li><li> 다시 잘 당 RPMI 1640 0.3 mL를 (페놀 레드없이)을 추가합니다. </li><li> 자기편 호중구 형광 (POST 세척) 단계 4.4에서뿐만 아니라 각각의 형광 강도를 측정한다. </li><li> 퍼센트 준수하고 잘 당 기준 준수를 확인합니다 : </li></ol> 참고 :이 단계에서, calcein - 라벨 호중구의 이미지가 표준 형광 필터 장착 된 형광 현미경 능력을 사용하여 얻을 수 있습니다. 그림 4의 이미지는 Pro7 소프트웨어 (Q 이미징) Q는 캡처를 사용 Retiga 2000R 카메라 (Q 영상)를 탑재 올림푸스 IX51 도립 현미경 하였다.

<ol>
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Immunol. 7, 803-815 (2007).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> J. Vis. Exp. (17), e745 (2008).</li><li>Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neutrophil+chemotaxis.">Analysis of neutrophil chemotaxis.</a> Methods Mol. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+1+acts+on+cultured+human+vascular+endothelium+to+increase+the+adhesion+of+polymorphonuclear+leukocytes,+monocytes,+and+related+leukocyte+cell+lines.">Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.</a> The Journal of Clinical Investigation. 76, (1985).</li><li>Schleimer, R. P., Rutledge, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+human+vascular+endothelial+cells+acquire+adhesiveness+for+neutrophils+after+stimulation+with+interleukin+1,+endotoxin,+and+tumor-promoting+phorbol+diesters.">Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters.</a> J. Immunol. 136, 649-654 (1986).</li><li>Gamble, J. R., Harlan, J. M., Klebanoff, S. J., Vadas, 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=Stimulation+of+the+adherence+of+neutrophils+to+umbilical+vein+endothelium+by+human+recombinant+tumor+necrosis+factor.">Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., 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=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, 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=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., 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=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, 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=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., 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=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

저자는 공개 아무것도 없어.

성공적으로 호중구 / 미세 혈관 내피 세포 접착 분석을위한 가장 중요한 단계는 다음과 같습니다 : 1) 낮은 통로 번호 (&lt;10), 건강한 내피 세포의 사용, 낮은 밀도 (즉, &lt;5 × 10 6 세포에서 분리 된 호중구를 유지 2) / ㎖) 및 분리 2 시간 이내에 그들을 사용하여, 각각 3) 까다로운 calcein AM-라벨 호중구의 세척 및 호중구에서 calcein의 EC 오염과 손실을 최소화하기 위해 적시에 calcein AM-라?...

혈관 내피 세포는 혈액 순환과 직접 접촉에 있기 때문에, 그것은 유일하게 감염이나 부상시 신속한 염증 반응을 시작할 수 있습니다. 내피 세포 (내피) 보존 세균의 구성 요소와 위험 분자의 다양성을 인식하고 표현 패턴 인식 수용체, 그리고 TNFα와 같은 호스트에 염증 매개체에 대한 수용체. 이들 수용체의 활성화는 내피는 사이토 카인 (예를 들어, IL-6, IL-8, CXCL1 및 CCL2) 분비하고, 그들의 세포 표면 1에서 부착 분자 (예를 들어, E / P-셀렉틴, VCAM-1, ICAM-1)를 상향 조절을 유도 2. 이 분자는 모든 전염성 에이전트의 호스트를 삭제하고 조직 수선에게 3,4을 시작하기 위해 감염과 부상의 사이트에 백혈구의 국산화를 촉진한다. 감염 호중구 반응은 혈관 내피 세포와 응답 사이의 잘 조율 된 상호 작용을 포함 호중구. EC의 활성화에 따라, IL-8 분비 감염이나 부상 5,6의 사이트에 집으로 호중구 수있는 내피 세포에서 혈관 그라데이션을 형성하고 있습니다. E / P-셀렉틴를 중재 호중구 캡처 및 호중구 세포 표면에 glycomolecules 상대적으로 약한 연결을 통해 압 연입니다. 이러한 상호 작용은 그 동족 수용체 IL-8 바인딩과 함께, 강력한, 인테-매개 부착과 내피 세포 표면 7-10 호중구 결국 체포를 용이하게합니다. 체포 후 호중구가 직접 병원균을 제거 병원균의 확산을 방지하기 위해 호중구 세포 트랩을 생성, 상처 치유를 촉진하고 단핵구, 대 식세포 및 수지상 등의 백혈구를 모집하는 추가 요인을 풀어 감염의 특정 사이트에 혈관 밖으로 마이그레이션 할 수 있습니다 세포 11-17. microv에 호중구 부착을 정량 할 수있는 체외 방법은이 보고서에 설명ascular는 TNFα 호스트 염증 매개체에 의해 활성화 된 후 ECS. 이 분석은 내피의 활성화, 그리고 호중구을 평가하기 위해 설계되었습니다. 인간의 기본 호중구 먼저 밀도 기울기 분리를 사용하여 격리하고, 다음 calcein 아세 톡시 (AM)로 표시되어 있습니다. 살아있는 세포 가수 분해 calcein에서 에스 테라 제는 492-495 nm의 513-516 nm의 18의 배출 여기에 높은 형광 calcein 분자 오전. 찬란 - 라벨 호중구는 다음 EC 단층으로 배양되고, 비 부착 호중구는 이후에 제거됩니다. 나머지 바운드 호중구의 형광 그 형광 분광 광도계를 사용하여 측정하고, 잘 당 총 호중구 형광 입력의 비율로 계산됩니다. 이 방법은 분광 광도계에 사용되는 바인딩 calcein - 라벨 호중구가 직접 EC의 AC 읽어 더 많은 정성을 제공하는 형광 현미경을 사용하여 시각화 할 수있는 추가적인 장점이 있습니다tivati​​on. 이 분석은 정적 인 조건에서 수행되기 때문에, 호중구 부착 폭포에서 발생 만 매우 초기 이벤트가 부과됩니다. 이 TNFα 처리 인간의 폐 혈관 EC (HMVEC 폐)에 해당 호중구 준수 E-셀렉틴과의 상호 작용이 중단 될 때 단일 층이 대폭 감소를 표시하는 E-셀렉틴 차단 항체를 사용하여이 보고서에서 확인됩니다. TNFα에 더하여, 우리는 성공적으로 수신자 같은 수용체 1 / 2 작용제 펩티도 글리 칸과 연관된 지단백 (PAL), murein 지방 단백질 (MLP)와 Pam3Cys에 의해 인간 제대 정맥 EC (HUVEC) 활성화의 범위를 결정하기 위해이 분석을 사용했습니다 Pam3Cys 19,20으로 HMVEC 폐 활성화. 또한, 우리는 성공적으로이 방법론의 다양한 호환되는 제안, 키나제 억제제와 HMVEC 폐의 표면과 세포질 단백질의 RNAi를 매개 최저 한 후이 분석을 사용 생화학 screeninG의 분석 20. 요약하면,이 분석은 체외에서 염증 매개 물질에 의해 EC 활성화의 범위를 액세스 할 재현성, 더 많은 기능을 방법을 사용하기 쉽게 제공합니다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

혈관 내피 세포는 염증 반응에 중요한 역할을한다. 염증의 급성 단계 동안, 내피 세포 (내피)는 호스트 매개로 직접 보존 미생물 구성 요소 또는 호스트 파생 위험 분자에 의해 활성화됩니다. 활성화 된 내피 표현 사이토 카인, 케모카인 동원과 접착 분자는, 감염이나 부상의 사이트에서 백혈구를 활성화하고 유지합니다. 호중구 도착하는 최초의 백혈구이며, 두 세포의 표면에 존재하는 접착 분자의 다양한 통해 내피을 준수합니다. 호중구의 주요 기능은 직접 미생물의 위협을 제거하는 추가 요인의 출시를 통해 다른 백혈구의 채용을 촉진하고, 상처 복구를 시작하는 것입니다. 따라서 내피 세포에 자신의 모집 및 첨부 염증 반응의 개시에 중요한 단계입니다. 이 보고서에서, 우리는 사용하는 체외 호중구 접착 분석에 대해 설명합니다calcein 정적 조건에서 미세 혈관 내피 세포의 활성화 정도를 정량하는 인간의 기본 호중구 AM-레이블. 이 방법은 형광 분광 광도계로 정량 동일한 샘플을 직접 호중구 바인딩보다 질적 평가를 위해 형광 현미경을 사용하여 시각화 할 수있는 추가적인 장점이 있습니다.

호중구 바인딩 분석을 사용하여 신뢰성, 재현성 결과를 얻기 위해서는 그림 1 HMVEC 폐에 그림과 같이 건강과 미세 혈관 내피 세포의 confluency에이 분석의 날에 최적이다하는 것이 필수적이다. 또한, 낮은 통로 번호 혈관 내피는 (이하 9 구절 즉,)를 사용하는 것이 필수적이며, 따라서, 우리는 해동 2 주 이내에 모든 실험을 수행하는 것이 좋습니다. 호중구의 건강은 세포가 어떤 방?...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

감염의 사이트에서 활성화 된 내피 세포에 백혈구 준수는 호스트의 염증 반응의 중요한 구성 요소입니다. 수 있습니다 호중구 결합 분석은이 보고서에 설명<em&gt; 체외에서</em정적 조건에서 염증 매개 물질에 의해 활성화 된 내피 세포에 바인딩 인간의 기본 호중구의&gt; 정량.

정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

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JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

17.0K 조회수.  University of California, San Francisco. 감염의 사이트에서 활성화 된 내피 세포에 백혈구 준수는 호스트의 염증 반응의 중요한 구성 요소입니다. 수 있습니다 호중구 결합 분석은이 보고서에 설명 체외에서 정량.

비디오: Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

포유류의 상피 세포에 박테리아의 접착 시험 관내 분석

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

연구

면역학 및 감염병학

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

흐름 조건에서 인간 제대 정맥 내피 세포 및 호중구 윤회의 연구에서 이들의 사용 격리

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

<em&gt; 체외에서</em공부하기&gt; 공동 감염 모델<em&gt; Plasmodium falciparum</em인간의 기본 단핵구 파생 면역 세포에서&gt; - HIV-1 상호 작용

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

시험관 내 병원균 유도 호중구 트랜스 상피 이동을 평가하는 공동 문화 분석

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

전단 응력의 인간 간 사인 내피에서 조건에 백혈구 모집을 연구하는 흐름 접착 분석

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

인간 호중구 유량 용기 접착 분석

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T 림프구에있는 인테그린 활성화의 연구에 대한 정적 접착 분석

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

호중구 윤회에서 생리 흐름 중 내피 세포 - 세포 접합의 실시간 영상

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...