이 작품은 건강의 국립 연구소에서 보조금 (JC에 P30 HL101302 및 RO1 HL109439)와 미국 심장 협회 (JC에 SDG 5270005)에 의해 부분적으로 지원되었다. A. Barazia은 T32HL007829 NIH 훈련 보조금에 의해 지원되었다.

1. Intravital 현미경의 준비 (그림 1A) <ol><li> (125 MM NaCl, 4.5 MM KCl, 2.5 MM CaCl 2, 1 MM MgCl 2, 17 MM NaHCO 3, pH를 7.4) superfusion 버퍼를 준비합니다. </li><li> 37 버퍼의 온도 및 온도 제어 이불을 유지하기 위해 순환 물 목욕을 켜 ° C. 질소 가스 (5 % CO 2 질소와 균형)으로 버퍼를 폭기. </li><li> 현미경 시스템을 켜십시오 (셔터 람다 DG-4 고속 파장 변환기, 워크 스테이션 컴퓨터, 올림푸스 BX61W 현미경, MPC-200 멀티 속이는, 교전 규칙-200 무대 컨트롤러, 고속 카메라, 강화제). </li><li> 혈관 염증 모델의 경우 100 % 이색 성 거울을 사용합니다. 레이저 부상으로 혈전 형성을 유도하기 위해 50분의 50 %의 거울 100 % 미러를 교체하고 micropoint 레이저 어블 레이션 시스템을 켜십시오. </li></ol> 2. Intravital 현미경에 대한 Cremaster 근육의 준비 (그림 1B) <ol><li> 를 마취케타민 (125 밀리그램 / kg 체중 (BW))과 xylazine의 IP 주입 (12.5 밀리그램 / kg BW)으로 남성 마우스 (6-8주, 이전 C57BL / 6). 일리노이 기관 동물 케어 및 사용위원회의 대학은 모든 동물의 보호와 실험 절차를 승인했다. 혈관 염증 모델의 경우 (수술하기 전에 2-2.5 시간) 이전 이미지에 intrascrotally 마우스를 3 시간에 손쥐 TNF-α를 (250 μl 생리의 0.5 μg) 주입. </li><li> intravital 현미경 트레이에 37 ° C에서 온도 제어 담요 위에 마우스를 놓습니다. </li><li> 집게를 사용하여 목의 표면에 피부를 당겨, 최대 가위로 1cm에 수평으로 목 피부의 중간 선을 절개. </li><li> 부드럽게 무딘 가위를 사용하여 자궁 근육을 열고기도를 둘러싼 근육을 제거합니다. </li><li> 호흡 곤란을 제거하는 기관에 PE90 튜브를 Cannulate. </li><li> 부드럽게 목 근육의 왼쪽을 제거하고 주변 조직에서 경정맥 정맥을 분리. </li><li> C annu항체 및 추가 anesthetics의 주입은 왼쪽 경정맥 정맥에 늦게 PE10 튜브. </li><li> 부드럽게 꺼내 수평으로 scrotal 피부를 절개. 실험을하는 동안 조직의 무결성을 유지하려면 prewarmed 버퍼는 수술 분야를 통해 superfused되었습니다. </li><li> 한 forcep으로 낮은 복부에 눌러 조심스럽게 다른 forcep과 고환을 당겨. </li><li> testis 주변의 주변의 결합 조직을 제거합니다. </li><li> 수직 cremaster 근육을 절개하고 주변을 달아하여 intravital 현미경 트레이에 유리 coverslip 위에 근육을 평평하게. </li><li> 이전 데이터 수집에 안정화 할 수있는 근육에 10-20 분 소요됩니다. 30-45 μm의 직경 Cremaster의 근육 arterioles (혈전 형성을위한)과 정맥은 (혈관 염증에 대한) 우리의 연구에 선정되었습니다. </li><li> 마취 조건을 유지하기 위해 동일한 마취 목 cannulus을 통해 약 매 30 분 30-50 ML을 불러 일으킬. </li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 3. TNF-α 유도 혈관 염증에 대한 Intravital 현미경 <ol><li> intravital 현미경, 오픈 SlideBook 5.0 소프트웨어에 마우스를 배치 한 후에 적절한 광학 및 목표 (60X)을 설정하는 메뉴 바의 &quot;초점 창 &#39;을 클릭합니다. </li><li> Dylight을 불러 일으킬 488 - 복합 쥐 안티 - 마우스 CD42c (100 μl 생리의 0.1 μg / g BW)와 알렉사 형석 647-복합 쥐 안티 - 마우스 GR-1 항체 (100 μl 생리에서 0.05 μg / g BW) 목 cannulus을 통해 . </li><li> 메뉴 표시 줄로 이동하여 도구 모음에서 &quot;이미지 캡처&quot;를 클릭하십시오. </li><li> 새 메뉴가 나타납니다; &quot;이미지&quot;에서 높이의 &quot;460&quot;을 1x1로 &quot;빈 팩터&quot;를 선택하고 &#39;640 &quot;너비에합니다. </li><li> &quot;필터 설정&quot;에서 각 노출 시간 노출 및 설정하고자하는 채널에 대한 확인란을 선택합니다. 예를 들어, 오픈 채널 : 10 밀리 초, FITC 채널 : 50 밀리 초 및 Cy5 채널 : 50 밀리 초. </li><li> 시간 포인트와 간격의 수를 조정하려면 &quot;시간 경과 캡처 &#39;를 선택합니다. 정R 염증 모델은, 시간 포인트의 수는 200 밀리 초의 간격 (5 분 총 경과 시간)을 1,000-1,500으로 설정되어 있습니다. </li><li> 해당 채널에 대한 지정된 노출 시간에 단일 비행기 이미지를 볼 수 있습니다 &quot;테스트&quot;를 클릭하세요. 히스토그램은 충분한 강도 분포 (멀티 채널 캡처를 들어, 각 채널에 대해이 과정을 반복)을 보여줍니다까지 노출 시간 및 / 또는 현미경 빛을 조정합니다. </li><li> 일단 모든 매개 변수가 &quot;이미지 캡처&quot;에 (채널, 노출 시간 등) 설정, 캡처를 시작하려면 &quot;시작&quot;을 선택합니다. </li><li> 혈관 염증 모델에서 염증 cremaster의 세정맥의 상단에 5 분을위한 압연 및 점착성의 혈소판과 호중구를 모니터링합니다. 8 10 개 정맥은 한 마우스에 기록됩니다. </li><li> 이미지는 157 μm X 118 μm의 창 크기를 제공 60X의 총 배율을 (1.0 NA 물 침적 목표)를 사용하여 촬영 된 것입니다. </li><li> 실험이 완료되면, 마우스는 자궁 경부 dislocatio 통해 euthanized되었다N. </li></ol> 4. Venular 염증 모델에 대한 데이터 분석 <ol><li> SlideBook 5.0 소프트웨어를 열고 파일을 열려면 메뉴 바에서 &quot;폴더&quot;를 클릭하십시오. </li><li> 드롭 다운 채널 메뉴를 클릭하고 &quot;열기, FITC, Cy5 등&quot;을 선택 </li><li> &quot;(빨간색과 초록색 막대) Renormalize&quot;와 채널을 선택을 클릭합니다. 약 형광 신호를 최적화 할 왼쪽 또는 오른쪽으로 빨간색 막대와 녹색 막대를 드래그합니다. </li><li> &quot;적용&quot;및 &quot;확인&quot;을 이미지를 업데이트를 클릭합니다. </li><li> 기본값으로 현재 디스플레이를 선택하려면 &#39;미리보기&#39;버튼을 클릭합니다. </li><li> 캡처 timelapse 이미지를 재생하고, 5 분 기간 동안 압연과 점착성의 호중구를 계산합니다. </li><li> 혈소판 혈전 형성을 분석하고, &quot;마스크&quot;로 이동하여 &quot;만들기&quot;를 선택합니다. </li><li> 마스크와 &quot;현재 이미지에서&quot;마크를 이름을 지정합니다. </li><li> 기본보기에서 &quot;움직이는 텍스트 도구&quot;에서 &quot;대형 연필 아이콘 &#39;을 클릭합니다. </li><li> 배경 마스크를 설정하는 기본보기에서 용기 외부 영역을 색상입니다. </li><li> t로 이동O &quot;마스크&quot;, &quot;비행기를 복사&quot;, &quot;현재 timepoint에 복사 마스크&quot;를 클릭하고 &quot;모든 timepoints&quot;을 선택 클릭하고 &quot;OK&quot;를 클릭하십시오. </li><li> 배경 신호를 계산하기 위해 &#39;통계&#39;로 이동 한 다음 &quot;마스크 통계 &#39;를 클릭합니다. </li><li> 이미지 범위에서 &quot;현재 2D 시간 경과 나 4D 이미지 (들)&quot;을 클릭하고 마스크 범위에서 &quot;전체 마스크&quot;를 선택합니다. 캡처 날짜에서 &quot;지역 (픽셀)&quot;Morphometry 아래 강도에 따라 &quot;최대 강도&quot;를 선택 선택하고 &quot;내보내기&quot;(이 통계 방법을 누릅니다 우리가 수행 할 수 있습니다 기능에서 &quot;경과 시간은 (: mm MS HH)&quot;를 선택 자동 형광 강도를 (배경 신호)) 계산합니다. </li><li> MS 오피스 Excel에서 텍스트 파일을 열고 녹음 기간 내내 배경 신호의 평균 값을 계산합니다. 이 배경 형광 강도입니다. </li><li> 혈전 강도를 확인하려면 기본보기에서 &quot;움직이는 텍스트 도구&quot;에서 다시 &quot;대형 연필 아이콘 &#39;을 클릭합니다. </li><li> 혈소판 신호를 설정하는 기본보기에서 용기 내부의 색상. </li><li> &#39;마스크&#39;로 이동, &quot;이 비행기를 복사합니다&quot;, &quot;모든 timepoints&quot;를 선택 &quot;현재 timepoint에 복사 마스크&quot;를 클릭하고 &quot;확인&quot;을 누릅니다. </li><li> &#39;통계&#39;로 이동하여 &#39;마스크 통계&#39;를 클릭합니다. </li><li> 이미지 범위에서 &quot;현재 2D 시간 경과 나 4D 이미지 (들)&quot;을 클릭하고 마스크 범위에서 &quot;전체 마스크&quot;를 선택합니다. 기능에서 선택 &quot;경과 시간 (HH : MM : 밀리 초)&quot;캡처 날짜에 따라, 우리에게 허용 &quot;지역 (픽셀)&quot;Morphometry 아래 강도에서 &quot;합 강도&quot;를 선택하고 &quot;내보내기&quot;(이 통계 방법을 클릭하고 ) 용기 내부의 형광 강도를 계산합니다. </li><li> MS 오피스 엑셀에서 텍스트 파일을 엽니 다. 혈소판의 형광 신호는 용기의 내부의 합 강도의 배경 농도 X 지역의 평균 값 (픽셀)을 차감하여 계산됩니다. 이것은 혈소판 혈전의 형광 강도입니다. </li><li> 3-5 다른 쥐 30 개 정맥에서이 작업을 반복합니다. 그런 다음, 혈소판의 형광 강도의 중간 값을 계산합니다. </li><li> 에호중구 압연과 접착력을 분석, 호중구의 압연 유입는 각 세정맥에 5 분 이상 결정과 분 당 전화 번호로 제시했다. &gt; 30 초 동안 정지했다 천천히 크롤링 (&lt;10 μm 30 초)하지만 이월 안 자기편 호중구의 수는 계산하고 5 분마다 전화 번호로 제시했다. </li></ol> 5. 레이저 유도 Arteriolar 혈전증에 대한 Intravital 현미경 절제 레이저의 5-1 보정 <ol><li> 현미경 단계에 미러링 된 슬라이드를 삽입, 슬라이드의 상단에 증류수의 작은 방울을 적용하고, 강도를 조정하고 접안 렌즈를 사용하여 초점을 맞 춥니 다. </li><li> &quot;초점 창&quot;을 열고 SlideBook 5.0 소프트웨어의 &#39;FRAP&#39;탭을 선택합니다. </li><li> 40-50에서 레이저 전원을 설정하고 모두 &quot;더블 클릭 반복&quot;과 &quot;더블 클릭 크기&quot;8로 설정합니다. 십자선의 설정을 가져 &quot;가이드&quot;상자에 표시합니다. </li><li> 작업 표시 줄 O에서 &#39;화살표&#39;아이콘을 선택합니다N 화면 상단. </li><li> &quot;FRAP 정렬&quot;탭에서 &quot;다음 불&quot;을 클릭하십시오. 작은 지점이 모니터의 왼쪽 하단 근처에 나타납니다. 그 자리가 표시되면 더블의 중심을 클릭합니다. 일단 클릭, 다른 장소 위 표시하고 첫 번째 지점의 오른쪽에해야한다, 그 위에 두 번 클릭합니다. 16 포인트 (16 일 현장의 오른쪽 하단 모서리에 나타납니다.) 보정 매개 변수를 업데이트 할 때 완료 &quot;저장&quot;을 클릭에 대해이 과정을 반복합니다. </li><li> 교정의 정확성을 테스트하려면, 화면의 중앙에 나타납니다 &quot;센터&quot;버튼 - 작은 곳을 클릭합니다. 그렇지 않을 경우 원하는 정밀도가 달성 될 때까지 보정을 반복합니다. </li></ol> 5-2 레이저 유도 arteriolar 혈전 형성 <ol><li> 현미경 단계에 anesthetized 마우스를 (2-2에 2-12의 절차를 참조) 놓습니다. </li><li> &quot;캡처&quot;창에서 프로토콜을 선택하거나 새 계정을 만드십시오. 설정은 다음과 같습니다 CY5 (70 밀리 초 노출), 오픈 (10 MSEC 노출) 및 FITC (50 밀리 초 노출). 이미지가 20 분 (500 밀리 초의 간격 시간이 약 2400 시간 점)을 통해 캡처됩니다. </li><li> Dylight 위에서 설명한 488-복합 항 마우스 CD42c와 알렉사 형석 647-복합 항 마우스 할머니-1 항체을 불러 일으킬. </li><li> 로 3-7으로 3-3에 설명 된 형광 강도 밝은 현장 이미지를 포함하는 현미경 매개 변수를 최적화합니다. </li><li> 적절한 brightfield 강도 또는 항체 신호, arteriole 게재 위치 및 초점을 보장하기 위해 &quot;테스트&quot;버튼을 클릭하십시오. </li><li> 이미지 캡처 프로세스를 시작하려면 &quot;시작&quot;을 클릭합니다. </li><li> 절제 레이저를 발사하기 전에 마우스 포인터 아이콘을 선택하고 혈관 벽이 명확한 초점에있는 것을되었는지 확인하십시오. </li><li> 레이저를 발사하려면 두 번 혈관 벽 2-3 μm 내부적으로 장소를 클릭하십시오. arteriolar 내피 세포의 손상은 brightfield 이미지에 명백해야 시각적 인 형상 변화가 발생, 혈소판 축적이 나타납니다. 모니터 혈전레이저 부상 후 5 분에 형성. </li><li> 레이저 부상 후 5 분 일시 정지 (혈전 크기가 지금은 안정적이어야합니다) 및 캡처를 취소합니다. 그 후, 20 분에 점착성의 혈소판 및 압연 / 점착성의 호중구를 기록 할 수있는 500 밀리 초 간격으로 2,400 시간을 포인트로 캡처를 시작합니다. 3 ~ 5 arterioles 한 마우스에 기록됩니다. </li><li> 실험이 완료되면, 마우스는 자궁 경부 전위를 통해 euthanized되었다. </li></ol> 6. Arteriolar 혈전증 모델에 대한 데이터 분석 <ol><li> 4.5 단계 4.1 이상 이동합니다. </li><li> 혈소판 혈전 형성하는 동안 배경 형광 강도를 구하려면 4.14 단계 4.7 이상 이동합니다. </li><li> &#39;마스크&#39;로 이동하여 &#39;세그먼트&#39;, 채널의 선택 FITC을 클릭하고 낮은에 배경 신호의 평균 값을 삽입합니다. 클릭하면 &quot;적용&quot;및 &quot;확인&quot;을. </li><li> 혈소판 혈전의 형광 강도 (Dylight 488-복합 항 CD42c 항체)를 분석하기 위해, </st롱은&gt; 4.19 단계 4.18으로 이동합니다. 항체 신호를 계산하려면, &quot;합 강도는&quot;( &quot;최대 강도&quot;X &quot;면적 (픽셀)&quot;) 배경 신호가 차감됩니다. 이것은 혈소판 혈전의 형광 강도입니다. </li><li> 압연과 점착성의 호중구을 수량화하려면 timelapse을 재생하고 가시적 20 분에 걸쳐 혈소판 혈전 위에 마우스를 셀의 개수를 계산합니다. 최소한 2 초 동안 혈소판 혈전과 상호 작용하는 동안 롤링은 호중구 속도 감소로 정의됩니다. 점착성의 호중구은 적어도 2 분 동안 혈소판 혈전에 연결된 상태로 유지하는 호중구로 정의됩니다. 압연과 점착성의 호중구의 수는 20 분 당 전화 번호로 제시했다. 롤링 속도는 입자 추적 시스템을 사용하여 계산되었습니다. </li></ol>

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R., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+extracellular+protein+disulfide+isomerase+during+thrombus+formation+in+mice.">A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.</a> J. Clin. Invest. 118, 1123-1131 (2008).</li><li>Cho, 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=Protein+disulfide+isomerase+capture+during+thrombus+formation+in+vivo+depends+on+the+presence+of+beta3+integrins.">Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.</a> Blood. 120, 647-655 (2012).</li><li>Gross, P. L., Furie, B. C., Merrill-Skoloff, G., Chou, J., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-versus+microparticle-mediated+tissue+factor+transfer+during+arteriolar+thrombus+development.">Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.</a> Journal of Leukocyte Biology. 78, 1318-1326 (2005).</li><li>Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+platelets,+tissue+factor+and+fibrin+during+arterial+thrombus+formation+in+the+mouse.">Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.</a> Nat. Med. 8, 1175-1181 (2002).</li><li>Barthel, S. 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=Alpha+1,3+fucosyltransferases+are+master+regulators+of+prostate+cancer+cell+trafficking.">Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 19491-19496 (2009).</li><li>Trzpis, M., McLaughlin, P. M., de Leij, L. M., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule:+more+than+a+carcinoma+marker+and+adhesion+molecule.">Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.</a> The American Journal of Pathology. 171, 386-395 (2007).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Egan, C. E., Sukhumavasi, W., Bierly, A. L., Denkers, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

저자는 경쟁 금융이자를 선언하지 않습니다.

여기 혈관 염증과 혈전증 동안 활성화 내피에 heterotypic 혈소판 호중구 상호 작용을 시각화하는 실시간 형광 intravital 현미경에 대한 자세한 프로토콜을 설명합니다. 이전 유사한 형광 현미경 방법은 혈전 형성과 혈관 염증의 분자 메커니즘을 연구보고되었다. 8,12가 heterotypic 세포 세포의 상호 작용은 부상 사이트에 vaso-가림을 위해 중요 할 수 있기 때문에,이 기술은를위한 가치있는 도구가...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

arteriolar 부상의 사이트에서 혈전 형성을하는 동안 활성화 된 내피의 mediates 혈전증과 혈관 염증에 활성화 된 혈소판 및 백혈구 (주로 호중구)의 상호 작용. 1,2, 활성화 내피와 subendothelial 매트릭스 단백질 자기편 혈소판은 호중구 압연과 접착력을 지원합니다. 3 반대로, venular 염증성 조건 하에서, 활성화 내피에 점착성의 호중구는 혈소판을 순환의 접착력과 축적을 지원할 수 있습니다. Heterotypic 혈소판 호중구 집계는 세포 사이의 특정 수용체 카운터 수용체의 상호 작용에 의해 순차적 프로세스를 필요로합니다. 4 그것은이 활성화 내피 세포는 따라서 높은 전단 조건 하에서 혈소판 부착 및 축적을 시작, 같은 폰 Willebrand 인자로 접착 분자를 풀어 알려져 있습니다. 5 또한, 활성화 내피 세포는 표현 selectins에 의한 호중구의 압연과 접착력을 지원D 세포 유착 분자 -1 (ICAM-1), 각각 낮은 전단 조건 하에서 4. 혈소판 P-selectin이 통과 호중구와 상호 작용하는 P-selectin 당 단백질 리간드-1 (PSGL-1),이를 호중구 β2 integrins 및 회사의 활성화를 유도 두 세포 유형 간의 접착. heterotypic 혈소판 호중구 상호 작용이 전체 혈액 또는 절연 세포, 6,7에서 결정되는 체외 실험에서의 발전에도 불구하고 그 연구는 혈관 질환 중 산화제 스트레스 조건을 조작 할 수 없습니다. 이 보고서에서, 혈소판 및 호중구 마커 마우스에 대한 휘황 라벨이 지정된 특정 항체를 사용하여, 우리는 TNF-α 유도 염증이나 레이저 유도에 따라하는 동안 활성화 된 내피에 혈소판과 호중구의 heterotypic 상호 작용을 모니터링 할 수있는 자세한 intravital 미세한 프로토콜을 설명 라이브 쥐 cremaster 근육 microvessels 부상.

자세한 intravital 현미경 분석을 사용하여 활성화 된 내피에 heterotypic 혈소판 호중구 상호 작용은 혈소판 (CD42c) 또는 호중구 마커 (GR-1) 라이브 생쥐에 대한 휘황-라벨 항체의 주입으로 시각화했다. TNF-α 유도 venular 염증 모델에서 대부분의 압연 호중구는 안정적으로 녹화 기간 (3-4.5 시간 TNF-α의 주입 후, 그림 2A 동안 ICAM-1 활성 β2 integrins의 상호 작용에 의해 아마 ?...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

여기 혈관 염증 및 라이브 쥐 혈전 형성되는 동안 활성화 내피에 heterotypic 혈소판 호중구 상호 작용을 시각화하는 형광 intravital 현미경의 실험 기술을보고합니다. 이 현미경 기술은 혈관 질환의 분자 메커니즘을 연구 할 수 있고 pathophysiological 조건에 따라 pharmacologic 요원을 테스트 할 가치가 있습니다.

라이브 마우스의 혈관 염증과 혈전 형성하는 동안 활성화 내피에 Heterotypic 혈소판 - 호중구 상호 작용의 실시간 영상

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K 조회수.  University of Illinois at Chicago. 여기 혈관 염증 및 라이브 쥐 혈전 형성되는 동안 활성화 내피에 heterotypic 혈소판 호중구 상호 작용을 시각화하는 형광 intravital 현미경의 실험 기술을보고합니다. 이 현미경 기술은 혈관 질환의 분자 메커니즘을 연구 할 수 있고 pathophysiological 조건에 따라 pharmacologic 요원을 테스트 할 가치가 있습니다.

비디오: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Leukotriene B의 실시간 영상 4 중재 셀 마이 그 레이션 및 β - arrestin로 BLT1 상호 작용

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

연구

면역학 및 감염병학

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

하이 Intraluminal 압력에서 혈관 내피에 이미징 백혈구의 접착

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

일반 CNS의 실시간 PCR에 의한과 Neuroinflammation 동안 Microglia에 MicroRNAs의 탐지

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T 세포의 실시간 라이브 영상은 복잡한 형성을 신호

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

소설<em&gt; 시험관</em&gt; 인간의 전체 혈액과 내피 세포 사이의 상호 작용 유학을위한 모델

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

영상 병인과의 Cording를 해독하고<em&gt; 미코 농양</em&gt; Zebrafish의 배아에서

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

실시간 세포 외 유출 분석을 사용하여 편광 M1과 M2 골수 유래 대 식세포의 대사 특성

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

시험관 에서 미세 유체 질환 모델 전체 혈액 내피 상호 작용 및 혈전 역학을 실시간으로 연구

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...