이 연구는 NIH NHLBI R35HL155657(N.M.)과 John C. Parker 교수직(N.M.)의 지원을 받았습니다. 도움이 되는 의견을 주신 Ms. Sierra J. Archibald에게 감사드립니다

이 연구는 노스캐롤라이나 대학교 채플힐(University of North Carolina at Chapel Hill)의 기관 검토 위원회(Institutional Review Board)의 승인을 받았습니다(프로토콜 번호: 14-2108).
1. 헌혈자의 혈액 채취
<ol><li>깨끗한 정맥 천자를 사용하여 21G 바늘로 전방 정맥에 전혈을 채취합니다. 혈액의 처음 3mL는 혈관 주위 세포의 TF를 포함할 수 있으므로 폐기하십시오.</li>
	<li>2.7 또는 1.8mL(튜브 크기에 따라 다름)의 혈액을 3.2% 구연산나트륨(0.109mol/L)이 포함된 바큐테이너에 넣습니다. 튜브를 과도하게 채우거나 부족하게 채우지 마십시오. 채혈 직후 튜브를 부드럽게 뒤집어 시트르산나트륨을 분산시킵니다.</li>
	<li>가공 전에 튜브를 운반하는 경우 교반을 피하십시오. 채혈 후 2시간 이내에 혈장을 준비합니다.</li>
</ol>2. 네거티브 대조군 혈장의 제조
알림: samples는 최종 동결 전에 얼음 위에 놓아서는 안 됩니다.
<ol><li>건강한 지원자의 전혈을 사용하여 음성 대조 혈장을 준비합니다. 채혈 직후 다음과 같이 무혈소판 혈장을 준비합니다.<ol><li>혈액 샘플을 vacutainer 튜브에서 15mL 튜브로 옮깁니다.</li>
		<li>브레이크 없이 실온(20-24°C)에서 15분 동안 2,500× g 에서 혈액 샘플을 원심분리합니다.</li>
		<li>상청액(혈소판 부족 혈장)을 새 튜브로 옮기고 동일한 조건에서 스핀을 반복합니다.</li>
		<li>상층액(혈소판 고갈 혈장)을 새 튜브로 옮깁니다.</li>
		<li>샘플을 ≥100 μL 부분 표본으로 분취합니다. 튜브 바닥에 약 100μL를 남겨 둡니다.</li>
		<li>혈소판이 고갈된 혈장을 -80°C에서 즉시 동결합니다(그림 1).</li>
	</ol></li>
</ol>3. 양성 대조 혈장의 제조
참고: 지질다당류에 대한 반응은 개인마다 다릅니다.
<ol><li>혈액 샘플을 vacutainer에서 15mL 튜브로 옮깁니다.</li>
	<li>대장균 O111:B4(10μg/mL)의 지질다당류(LPS)로 자극된 건강한 지원자의 전혈을 사용하여 37°C에서 5시간 동안 교반하면서 양성 대조 혈장을 준비합니다.</li>
	<li>5시간 배양 후 2.1.2에서 2.1.6 단계를 따르십시오.</li>
</ol>4. 혈장에서 세포외 소포체의 분리
알림: EV 펠릿이 보이지 않을 수 있습니다. 해동 시간은 시료량에 따라 다르지만 일반적으로 37°C에서 30분 동안 100μL 혈장을 해동합니다.
<ol><li>혈장 시료를 37°C에서 해동합니다.</li>
	<li>1.5mL 튜브에 있는 각 100μL의 혈장 샘플에 칼슘[HBSA-Ca(-)] 완충액[137mM NaCl, 5.38mM KCl, 5.55mM 포도당, 10mM HEPES, 0.1%(w/v) 소 혈청 알부민]이 없는 HBSA 1mL를 추가합니다.</li>
	<li>플라즈마 시료를 20,000 × g 에서 4 °C에서 15분 동안 원심분리합니다.</li>
	<li>EV 펠릿을 방해하지 않고 상층액을 20μL까지 흡입합니다.</li>
	<li>각 튜브에 HBSA-Ca(-) 완충액 1mL를 첨가하여 EV 펠릿을 재구성하고, EV 펠릿 위치에서 피펫팅을 위아래로 하고, 각 튜브를 소용돌이친 후 4°C에서 15분 동안 20,000× g 에서 두 번째로 원심분리합니다.</li>
	<li>EV 펠릿을 방해하지 않고 상층액을 20μL까지 흡입합니다.</li>
	<li>80μL의 HBSA-Ca(-)에서 EV 펠릿을 재구성하고 EV 펠릿 위치에서 위아래로 피펫팅합니다(그림 2).</li>
</ol>5. 선택권: ultracentrifuge를 사용하여 작은 extracellular 소포의 고립
<ol><li>4.3단계 후 상층액과 초원심분리기를 100,000× g 에서 4°C에서 70분 동안 수집합니다.</li>
	<li>EV 펠릿을 방해하지 않고 상층액을 20μL까지 흡입합니다.</li>
	<li>각 튜브에 1mL의 HBSA-Ca(-) 완충액을 첨가하여 EV 펠릿을 재구성하고 각 튜브를 소용돌이친 후 4°C에서 70분 동안 100,000× g 에서 두 번째로 초원심분리합니다.</li>
	<li>EV 펠릿을 방해하지 않고 상층액을 20μL까지 흡입합니다.</li>
	<li>EV 펠릿을 80μL의 HBSA-Ca(-)로 재구성합니다.</li>
</ol>6. 세포외 소포체 조직 인자 활성 측정
알림: EV 샘플은 웰에 추가하기 전에 잘 피펫팅하고 볼텍싱하여 혼합해야 합니다. EDTA-디소듐은 FXa가 발색 기질을 절단하는 능력을 억제합니다. 따라서 EDTA-디소듐은 6.7단계에서 EDTA-테트라소듐의 대체품으로 사용할 수 없습니다.
<ol><li>40μL의 EV 샘플을 96웰 플레이트의 웰 2개에 추가합니다.</li>
	<li>11μL의 억제성 마우스 항인간 TF IgG[(36.4μg/mL, 최종 농도 7.8μg/mL)]를 EV 샘플의 한 웰에 추가하고 11μL의 대조군 마우스 IgG[(36.4μg/mL, 최종 농도 7.8μg/mL)]를 다른 웰에 추가합니다.</li>
	<li>실온에서 15분 동안 96웰 플레이트를 배양합니다.</li>
	<li>배양 시간 동안 50μL/웰의 TF 표준물질(0, 0.32, 0.63, 1.25, 2.5, 5, 10 및 20pg/mL)을 relipidated recombinant TF를 사용하여 중복으로 준비합니다.</li>
	<li>4mL의 HBSA를 칼슘[HBSA-Ca(+)][HBSA-Ca(-) + 10mMCaCl2, 최종 농도 10mM], HBSA-Ca(+)의 900nM FX 800μL(최종 농도: 146.4nM), HBSA-CA(+)의 200nM FVIIa 120μL(최종 농도: 4.8nM)와 혼합하여 인자 혼합물을 준비합니다.</li>
	<li>각 웰에 50μL의 인자 혼합 용액을 추가하고 플레이트를 필름으로 덮은 다음 37°C의 인큐베이터에서 2시간 동안 배양합니다.</li>
	<li>2시간 후 HBSA 에틸렌디아민테트라아세트산(EDTA) 완충액 25μL[HBSA-Ca(-) + 25mM EDTA-테트라소듐, 최종 농도 5mM]을 첨가하여 FXa 생성을 중지하고 실온에서 5분 동안 배양합니다.</li>
	<li>FXa에 의해 절단된 발색 기질을 4mM으로 재구성합니다(25mg 바이알에 증류수 8.7mL 추가).</li>
	<li>각 웰에 25μL의 발색 기질 용액을 추가하고 플레이트를 필름으로 덮은 다음 알루미늄 호일로 덮어 빛으로부터 보호하고 37°C에서 15분 동안 배양합니다.</li>
	<li>15분 배양 후 바늘로 기포를 제거하거나 1,500× g 에서 1분 동안 원심분리합니다.</li>
	<li>판독하기 전에 한 번 혼합된 플레이트 리더를 사용하여 405nm에서 플레이트를 판독합니다.</li>
	<li>relipidated recombinant TF를 사용하여 생성된 표준 곡선을 사용하여 각 웰의 FXa 생성을 TF 활성으로 변환합니다.</li>
	<li>방정식 (1)을 사용하여 TF 종속 FXa 생성(EVTF 활성)을 계산합니다. TF 종속 FXa 생성(EVTF 활성[pg/mL]) = 총 FXa 생성(제어 IgG 웰) - TF 독립적 FXa 생성(항 TF IgG 웰)(그림 3)(1)</li>
</ol>

세포외 소포체(Extracellular Vesicles)의 조직 인자 활성 분석(Tissue Factor Activity Assay)에 대한 감도 향상

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L., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+new+bead-based+assay+to+measure+levels+of+human+tissue+factor+antigen+in+extracellular+vesicles+in+plasma.">Evaluation of a new bead-based assay to measure levels of human tissue factor antigen in extracellular vesicles in plasma.</a> Research and Practice in Thrombosis and Haemostasis. 6 (2), e12677 (2022).</li><li>Tatsumi, K., 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=Evaluation+of+a+new+commercial+assay+to+measure+microparticle+tissue+factor+activity+in+plasma:+communication+from+the+SSC+of+the+ISTH.">Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH.</a> Journal of Thrombosis and Haemostasis. 12 (11), 1932-1934 (2014).</li><li>Hisada, 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=Measurement+of+microparticle+tissue+factor+activity+in+clinical+samples:+A+summary+of+two+tissue+factor-dependent+FXa+generation+assays.">Measurement of microparticle tissue factor activity in clinical samples: A summary of two tissue factor-dependent FXa generation assays.</a> Thrombosis Research. 139, 90-97 (2016).</li><li>Tatsumi, K., Hisada, Y., Connolly, A. F., Buranda, T., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+severe+orthohantavirus+cardiopulmonary+syndrome+due+to+Sin+Nombre+Virus+infection+have+increased+circulating+extracellular+vesicle+tissue+factor+and+an+activated+coagulation+system.">Patients with severe orthohantavirus cardiopulmonary syndrome due to Sin Nombre Virus infection have increased circulating extracellular vesicle tissue factor and an activated coagulation system.</a> Thrombosis Research. 179, 31-33 (2019).</li><li>Schmedes, C. 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=Circulating+Extracellular+Vesicle+Tissue+Factor+Activity+During+Orthohantavirus+Infection+Is+Associated+With+Intravascular+Coagulation.">Circulating Extracellular Vesicle Tissue Factor Activity During Orthohantavirus Infection Is Associated With Intravascular Coagulation.</a> Journal of Infectious Diseases. 222 (8), 1392-1399 (2020).</li><li>Rosell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+COVID-19+have+elevated+levels+of+circulating+extracellular+vesicle+tissue+factor+activity+that+is+associated+with+severity+and+mortality-Brief+report.">Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-Brief report.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 878-882 (2021).</li><li>Campbell, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+coagulopathies+associated+with+COVID-19+and+sepsis.">Comparison of the coagulopathies associated with COVID-19 and sepsis.</a> Research and Practice in Thrombosis and Haemostasis. 5 (4), e12525 (2021).</li><li>Guervilly, C., 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=Dissemination+of+extreme+levels+of+extracellular+vesicles:+tissue+factor+activity+in+patients+with+severe+COVID-19.">Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.</a> Blood Advances. 5 (3), 628-634 (2021).</li><li>Hisada, Y., Mackman, N. <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+tissue+factor+activity+in+extracellular+vesicles+from+human+plasma+samples.">Measurement of tissue factor activity in extracellular vesicles from human plasma samples.</a> Research and Practice in Thrombosis and Haemostasis. 3 (1), 44-48 (2019).</li><li>Sachetto, A. T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+factor+activity+of+small+and+large+extracellular+vesicles+in+different+diseases.">Tissue factor activity of small and large extracellular vesicles in different diseases.</a> Research and Practice in Thrombosis and Haemostasis. 7 (3), 100124 (2023).</li><li>Bom, V. J., Bertina, R. 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+contributions+of+Ca2+,+phospholipids+and+tissue-factor+apoprotein+to+the+activation+of+human+blood-coagulation+factor+X+by+activated+factor+VII.">The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.</a> Biochemical Journal. 265 (2), 327-336 (1990).</li><li>Tilley, R. E., Holscher, T., Belani, R., Nieva, J., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+factor+activity+is+increased+in+a+combined+platelet+and+microparticle+sample+from+cancer+patients.">Tissue factor activity is increased in a combined platelet and microparticle sample from cancer patients.</a> Thrombosis Research. 122 (5), 604-609 (2008).</li><li>Gurung, S., Perocheau, D., Touramanidou, L., Baruteau, 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+exosome+journey:+from+biogenesis+to+uptake+and+intracellular+signalling.">The exosome journey: from biogenesis to uptake and intracellular signalling.</a> Cell Communication and Signaling. 19 (1), 47 (2021).</li><li>Garnier, D., 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=Cancer+cells+induced+to+express+mesenchymal+phenotype+release+exosome-like+extracellular+vesicles+carrying+tissue+factor.">Cancer cells induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor.</a> Journal of Biological Chemistry. 287 (52), 43565-43572 (2012).</li><li>Park, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+factor-bearing+exosome+secretion+from+human+mechanically+stimulated+bronchial+epithelial+cells+in+vitro+and+in+vivo.">Tissue factor-bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo.</a> Journal of Allergy and Clinical Immunology. 130 (6), 1375-1383 (2012).</li></ol>

공개할 상충되는 이해관계가 없습니다.

여기에서는 자체 EVTF 활성 분석의 프로토콜이 제시됩니다. 프로토콜에는 세 가지 중요한 단계가 있습니다. EV 펠릿을 재구성할 때, EV 펠릿이 보이지 않더라도 EV 펠릿의 위치에서 위아래로 피펫팅하는 것이 중요합니다. EV 펠릿의 불완전한 재구성은 샘플의 EVTF 활성 값의 위음성 또는 과소 평가를 초래합니다. 둘째, 칼슘 없이는 FXa 생성이 불가능하기 때문에 프로토콜 단계 6.5에서 HBSA-Ca(+)를 사용하?...

혈액 응고는 인자 (F) VII/VIIa와 조직 인자(TF)1의 결합으로 시작됩니다. TF/FVIIa 복합체는 FIX와 FX를 모두 활성화하여 혈액 응고를 활성화한다1. 전체 길이, 멤브레인 결합 TF에는 암호화와 활성의 두 가지 형태가 있습니다. 또한, 대안적으로 접합된 형태의 TF(asTF)가 있습니다. 세포막의 바깥쪽 엽단에 있는 스핑고미엘린과 포스파티딜콜린은 TF를 암호화된 상태로 유지합니다 2,3,4. 세포가 활성화되거나 손상되면 인지질 스크램블라제는 포스파티딜세린 및 기타 음전하를 띤 인지질을 바깥쪽 전단으로 전달합니다1. 세포의 활성화는 또한 산성 스핑고미엘라아제를 외부 전단지로 전위시켜 스핑고미엘린을 세라마이드로 분해합니다5. 이 두 메커니즘은 암호화된 TF를 활성 형식으로 변환합니다. 또한 단백질 이황화 이소머라제가 암호화된 TF에서 Cys186과 Cys209 사이의 이황화 결합 형성을 매개하여 TF 6,7,8의 해독을 초래하는 것으로 제안되었습니다. asTF는 순환에도 존재하지만 막관통 도메인이 부족하므로 용해성 9,10입니다. 중요한 것은 asTF가 전장 활성 TF10,11에 비해 매우 낮은 수준의 응고제 활성을 가지고 있다는 것입니다.
세포외 소포체(EV)는 암세포뿐만 아니라 휴지기, 활성화 및 죽어가는 숙주 세포에서 방출된다12. EV는 부모 세포에서 단백질을 발현한다12. 활성 TF 함유 EV는 활성화된 단핵구, 내피 세포 및 종양 세포로부터 순환 13,14,15로 방출된다. 혈장 내 TF 수치는 활성 및 항원 기반 분석으로 측정할 수 있습니다. 항원 기반 분석에는 ELISA 및 유세포 분석이 포함됩니다16. 활동 기반 분석에는 1단계 및 2단계 TF 활동 분석의 두 가지가 있습니다. 1단계 분석은 혈장 기반 응고 분석을 기반으로 합니다. TF 함유 샘플을 혈장에 첨가하고 재석회화 후 응고를 형성하는 시간을 측정합니다. 2단계 분석은 FVII 또는 FVIIa, FX 및 칼슘을 추가하여 샘플의 FXa 생성을 측정합니다. FXa 레벨은 FXa에 의해 절단된 기판을 사용하여 결정됩니다.
1단계 및 2단계 TF 활성 분석 모두에서 TF 농도는 재조합 TF로 생성된 표준 곡선을 사용하여 측정됩니다. 2단계 분석은 1단계 분석보다 민감도와 특이도가 높습니다. 많은 연구에서 활성 기반 분석이 항원 기반 분석보다 민감도와 특이도가 더 높다는 것을 확인했습니다 17,18,19,20,21. 또한, 당사의 자체 활성 분석은 상용 활성 분석보다 더 높은 민감도와 특이성을 가지고있습니다 22. 건강한 사람은 혈장에서 EVTF 활성이 매우 낮거나 검출할 수 없는 수준입니다. 대조적으로, 암, 간경변, 패혈증 및 바이러스 감염과 같은 병리학적 상태가 있는 개인은 EVTF 활성이 검출 가능한 수준이며 이는 혈전증, 파종성 혈관 내 응고, 질병 중증도 및 사망률과 관련이 있습니다 23,24,25,26,27,28. 여기에서는 이 사내 2단계 EVTF 활성 분석에 대해 설명합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube for 20,000 x g centrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Fisher Scientific (Catalog number: 05-408-129).</td>
		</tr>
		<tr>
			<td>1.5 mL tube for ultracentrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Beckman Coulter (Catalog number: 357448)</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from VWR (Catalog number: 89039-666)</td>
		</tr>
		<tr>
			<td>21 G x .75 in. BD Vacutainer Safety-Lok Blood Collection Set with 12 in. tubing and luer adapter</td>
			<td>BD</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Globe Scientific (Catalog number: 120338).</td>
		</tr>
		<tr>
			<td>BD Vacutainer Citrate Tubes</td>
			<td>BD</td>
			<td>363083</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5417R (Eppendorf).</td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5810R (Eppendorf).</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FVIIa</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFVIIa</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Human FX</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFX1010</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Inhibitory mouse anti-human tissue factor IgG, clone HTF-1</td>
			<td>Fisher Scientific</td>
			<td>550252</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Escherichia coli O111:B4</td>
			<td>Sigma Aldrich</td>
			<td>L2630</td>
			<td>There are several lipopolysaccharide from different E. coli. Different lipopolysaccharide have different potential to activate monocytes.</td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Sigma Aldrich</td>
			<td>I5381</td>
			<td></td>
		</tr>
		<tr>
			<td>Pefachrome FXa 8595</td>
			<td>Enzyme Research Laboratory</td>
			<td>085-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the SpectraMax i3x from Molecular Devices</td>
		</tr>
		<tr>
			<td>Re-lipidated recombinant tissue factor, Dade Innovin</td>
			<td>Siemens</td>
			<td>10873566</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima TLX</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>TLA-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

extracellular vesicle tissue factor activity assay

조직 인자(TF)는 인자 (F) VII 및 FVIIa에 대한 막관통 수용체입니다. TF/FVIIa 복합체는 FIX와 FX를 모두 활성화하여 응고 캐스케이드를 시작합니다. TF는 세포외 소포체(EV)의 형태로 세포에서 순환계로 방출됩니다. TF 양성(+) EV의 수준은 암, 박테리아 및 바이러스 감염, 간경변을 포함한 다양한 질병에서 증가하며 혈전증, 파종성 혈관 내 응고, 질병 중증도 및 사망률과 관련이 있습니다. 혈장에서 TF+ EV를 측정하는 방법에는 항원 기반 분석과 활성 기반 분석의 두 가지가 있습니다. 데이터에 따르면 활성 기반 분석은 항원 기반 분석보다 민감도와 특이도가 더 높습니다. 이 백서는 2단계 FXa 생성 분석을 기반으로 하는 당사의 사내 EVTF 활성 분석에 대해 설명합니다. FVIIa, FX 및 칼슘을 TF+ EV 함유 샘플에 첨가하여 항 TF 항체의 존재 유무에 따라 FXa를 생성하여 TF 의존성 FXa 생성과 TF 독립적 FXa 생성을 구별합니다. FXa에 의해 절단된 발색 기질은 FXa 수준을 결정하는 데 사용되며, TF 농도를 측정하기 위해 relipidated recombinant TF로 생성된 표준 곡선이 사용됩니다. 이 자체 EVTF 활성 분석은 상용 TF 활성 분석보다 민감도와 특이도가 높습니다.

성공적인 결과는 ≥0.5pg/mL의 양성 대조군과 <0.5pg/mL의 음성 대조군을 제공합니다. 양성 대조군을 위해 >1.0pg/mL EVTF 활성을 가진 높은 LPS 반응기를 찾는 것이 가장 좋습니다. 대표적인 결과는 LPS 활성화 유무에 관계없이 건강한 기증자 11명의 전혈에서 혈장에서 분리한 EV의 EVTF 활성을 보여줍니다(그림 4). 11명의 기증자 중 6명(기증자 2, 4, 5, 8, 10, 11명)은 중등도에서 높은 LPS 반응...

Watch this Scientific Journal Video about High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis at JoVE.com

저자 스포트라이트: 혈장 진단을 위한 고감도 조직 인자 활성 분석 

여기서는 사내 세포외 소포체 조직 인자 활성 분석에 대해 설명합니다. 활성 기반 분석 및 항원 기반 분석은 인간 혈장 샘플에서 세포외 소포체의 조직 인자를 측정하는 데 사용되었습니다. 활성 기반 분석은 항원 기반 분석보다 민감도와 특이도가 더 높습니다.

세포외 소포 조직 인자 활성 분석

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX ...

낮은 수준의 조직 인자 양성 세포외 소포체는 다양한 질병을 가진 환자의 혈장에 존재합니다. 그러나 ELISA 및 유세포 분석을 포함한 대부분의 항원 기반 분석은 이러한 낮은 수준을 검출할 수 있는 감도가 없습니다. 수많은 논문에서 혈장에서 조직 인자를 검출하기 위해 ELISA 또는 유세포 분석을 사용했습니다.당사의 프로토콜은 높은 감도와 특이성으로 혈장에서 조직 인자 활성을 측정하지만, 기능적 분석의 사용으로 인해 약간의 변동이 있습니다. 혈장에서 조직 인자 활성 수준을 측정하는 상용 분석 키트가 있습니다. 그러나 이러한 키트에는 항조직 인자 항체가 포함되어 있지 않으므로 조직 인자 의존적 및 조직 인자 독립적 Factor X 생성을 구별할 수 없습니다.

extracellular-vesicle-tissue-factor-activity-assay

Research

JoVE Journal

Medicine

Extracellular Vesicle Tissue Factor Activity Assay

664 조회수.  University of North Carolina at Chapel Hill. 낮은 수준의 조직 인자 양성 세포외 소포체는 다양한 질병을 가진 환자의 혈장에 존재합니다. 그러나 ELISA 및 유세포 분석을 포함한 대부분의 항원 기반 분석은 이러한 낮은 수준을 검출할 수 있는 감도가 없습니다. 수많은 논문에서 혈장에서 조직 인자를 검출하기 위해 ELISA 또는 유세포 분석을 사용했습니다.당사의 프로토콜은 높은 감도와 특이성으로 혈장에서 조직 ?...

비디오: 저자 스포트라이트: 혈장 진단을 위한 고감도 조직 인자 활성 분석 

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

실험 전이 분석

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

연구

의학

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

콜레스테롤 유출 분석

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

마우스 모델에서 하대 정맥 끼어 이식의 주입

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

공 초점 광학 이미징에 의해 로즈 벵갈 Photothrombosis<em&gt; 생체</em&gt; : 단일 선박 스트로크의 모델

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

전략적 내피 세포 관 형성의 분석 : 감소 세포 외 매트릭스 세포 외 기질과 성장 인자를 비교

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D 전체 심장 심근 조직 분석

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

조사 폰 Willebrand 요소 이상 모델을 사용 하는 흐름 챔버의 폰 Willebrand 요인-혈소판 문자열 형성

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

중증 환자에서 전혈 내독소 혈증 검출을 위한 내독소 활성 분석

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

비-침략 적 쥐에서 Dorsiflexor 근육 기능 평가

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...