이 작품은 낸시 Lurie는 마크 재단에 의해 지원되었다. 우리는 원고의 중요한 읽기 박사 낸시 E. 콜 감사합니다. 우리는이 비디오 보고서를 준비하고있는 그의 도움을 킨 K. 웡 감사합니다.

참고 : 모든 동물 실험이 승인 제도 프로토콜 및 동물 관리 지침에 따라 수행되었다. 1. 시약 및 솔루션 <ol><li> SC 접종에 대한 PMA 솔루션 : phorbol 12-스틴 산 5 MG / DMSO에 ML에서 13 아세테이트 (PMA)의 주식 솔루션을 준비합니다. -20 ° C.에 재고 솔루션을 저장 접종 전에 재고 솔루션을 해동하고 PBS 1 MG / ML PMA에 희석. </li><li> SC 접종에 대한 LPS 솔루션 : 이전 SC 접종 1 MG / ML에서 멸균 PBS에 용해 리포 다당류 (살모넬라 균의 enterica 혈청 형 장염에서 LPS). </li><li> 급성기 이미징 루미놀 용액 : 10 ㎎ / ㎖에서 (0.9 % 염화나트륨) 멸균 생리 식염수에 (5 - 아미노 -2,3 - 디 하이드로 -1,4 - phthalazinedione, 나트륨 염) 루미놀을 녹인다. 이 솔루션은 사용하기 전에 -20 ° C에 저장할 수 있습니다. </li><li> 만성 단계 이미징 Lucigenin 솔루션 : 멸균 생리 식염수에 lucigenin (BIS-N-methylacridinium 질산)를 녹여2.5 MG / ML. 이 솔루션은 사용하기 전에 -20 ° C에 저장할 수 있습니다. </li></ol> 2. 피하 PMA 염증 모델 <ol><li> 2 % isoflurane을 가진 유도 챔버에있는 NCR 누드 마우스를 마취. 운동의 손실과 일정한 호흡 속도에 의해 전신 마취를 확인합니다. isoflurane을 혼합 가스를 공급 코 콘에 동물을 전송하고 마취하에 동물을 유지합니다. </li><li> 왼쪽 측면에 주사 부위를 청소하고 소독 닦아 이소 프로필 알코올을 사용합니다. </li><li> 무균 기술을 사용하여, 왼쪽 측면에 피하 공간으로 PMA 접종 용액 (PMA의 50 μg을 포함)의 50 μl를 주입. 이소 프로필 알코올 패드를 사용하여 주사 부위에서 초과 액체를 제거합니다. 이 염증 반응에 영향을 미칠 수 있으므로 진통제의 사용을 피하십시오. </li><li> 주택 케이지로 동물을 이동하고 밀접하게 마취로부터 회복을 모니터링 할 수 있습니다. 복구를 지원하기 위해, 동물을 따뜻하게 유지하기 위해 가열 패드를 사용합니다.</li></ol> 3. 피하 LPS 염증 모델 <ol><li> 유도 챔버에서 isoflurane을 1 ~ 2 %로 C57BL/6J 마우스를 마취. 전신 마취를 설정하면, isoflurane을 가스 공급 노즈 콘에 동물을 전송하고 마취하에 동물을 유지합니다. </li><li> 왼쪽 발바닥에 주사 부위를 청소 닦아 이소 프로필 알코올을 사용합니다. </li><li> 무균 기술을 사용하여, 왼쪽 발바닥에 LPS 접종 용액 (LPS의 50 μg을 포함)의 50 μl를 주입. 이소 프로필 알코올 패드를 사용하여 주사 부위에서 초과 액체를 제거합니다. 이 염증 반응에 영향을 미칠 수 있으므로 진통제의 사용을 피하십시오. </li><li> 주택 케이지로 동물을 이동하고 밀접하게 마취로부터 회복을 모니터링 할 수 있습니다. 복구하는 동안 동물을 따뜻하게 유지하기 위해 가열 패드를 사용합니다. </li></ol> 4. 염증의 생물 발광 이미징 <ol><li> 2 % isoflurane을 혼합 가스를 유도 챔버에서 동물을 마취. 공동운동의 손실과 일정한 호흡 속도에 의해 전신 마취를 nfirm. </li><li> 동물이 마취하에있는 동안, 복강 (IP)은 각각 급성 또는 만성 염증 이미징 루미놀 용액 (10 ㎎ / ㎖) 또는 lucigenin 솔루션 (2.5 MG / ML) 주입. 마지막 복용량은 루미놀과 lucigenin 25 ㎎ / ㎏ 100 ㎎ / ㎏이다. lucigenin (10 ~ 15 ㎎ / ㎏)의 낮은 복용량은 몇 마우스 종자 가능한 독성을 방지하는 데 사용할 수 있습니다. 독성의 징후는 호흡과 호흡 곤란 곤란이 (가) 있습니다. C57BL/6J 변형은 NCR 누드 변형보다 낮은 lucigenin 인내를 가지고 있습니다. </li><li> 생물 발광 영상 시스템의 이미징 챔버에 동물을 전송합니다. </li><li> 1 분 간격으로 순차적 생물 발광 영상을 수행합니다. 각 이미징 단계를 1 분 수집 시간으로 구성되어, F / 정지 = 1, 비닝 (binning) = 16 0 초 지연. </li><li> 이미지 수집 패널 내에서 순차적으로 여러 단계의 이미지를 사용하려면, BU를 설정하는 순서를 클릭하십시오tton. 최대 발광 출력 (보통 15 1 분 단계는 충분합니다)를 결정하기 위해 취득 프로필에 충분한 이미징 단계를 제공합니다. 15 분 영상 부분은 기판 흡수 및 전신 순환을 위해 충분한 시간을 허용합니다. </li><li> 이미징 챔버에서 동물을 제거하고 하우징 케이지로 다시 이동합니다. 복구를 지원하기 위해, 동물을 따뜻하게 유지하기 위해 가열 패드를 사용합니다. </li><li> 취득 후 분석 중에 관심의 표준화 영역 (ROI)를 통해 최대 총 발광 신호를 계산하는 이미징 소프트웨어 패키지를 사용합니다. 이미지 / 최소 및 최대 임계 값을 갖는 초 / cm 2 / SR이 표시 광자의 생기로 존재한다. 정량적 데이터는 ROI 당 초당 광자의 광속으로 표시됩니다. </li></ol>

<ol>
	<li>Premack, B. A., Schall, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+receptors:+gateways+to+inflammation+and+infection.">Chemokine receptors: gateways to inflammation and infection.</a> Nat. Med. 2, 1174-1178 (1996).</li><li>Singer, A. J., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing.">Cutaneous wound healing.</a> N. Engl. J. Med. 341, 738-746 (1999).</li><li>Wellen, K. E., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+stress,+and+diabetes.">Inflammation, stress, and diabetes.</a> J. Clin. Invest. 115, 1111-1119 (2005).</li><li>Weitzman, S. A., Gordon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+cancer:+role+of+phagocyte-generated+oxidants+in+carcinogenesis.">Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis.</a> Blood. 76, 655-663 (1990).</li><li>Ridker, P. M., Cushman, 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=Inflammation,+aspirin,+and+the+risk+of+cardiovascular+disease+in+apparently+healthy+men.">Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men.</a> N. Engl. J. Med. 336, 973-979 (1997).</li><li>Wyss-Coray, T., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+neurodegenerative+disease--a+double-edged+sword.">Inflammation in neurodegenerative disease--a double-edged sword.</a> Neuron. 35, 419-432 (2002).</li><li>Hamilton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-stimulating+factors+in+inflammation+and+autoimmunity.">Colony-stimulating factors in inflammation and autoimmunity.</a> Nat. Rev. Immunol. 8, 533-544 (2008).</li><li>Harlan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+interactions.">Leukocyte-endothelial interactions.</a> Blood. 65, 513-525 (1985).</li><li>Bedard, K., Krause, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOX+family+of+ROS-generating+NADPH+oxidases:+physiology+and+pathophysiology.">The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.</a> Physiol. Rev. 87, 245-313 (2007).</li><li>Rest, R. F., Spitznagel, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+distribution+of+superoxide+dismutases+in+human+neutrophils.+Influence+of+myeloperoxidase+on+the+measurement+of+superoxide+dismutase+activity.">Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity.</a> Biochem. J. 166, 145-153 (1977).</li><li>Hampton, M. B., Kettle, A. 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=Inside+the+neutrophil+phagosome:+oxidants,+myeloperoxidase,+and+bacterial+killing.">Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing.</a> Blood. 92, 3007-3017 (1998).</li><li>Serhan, C. N., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+the+beginning+programs+the+end.">Resolution of inflammation: the beginning programs the end.</a> Nat. Immunol. 6, 1191-1197 (2005).</li><li>Jiang, F., Zhang, 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=NADPH+oxidase-mediated+redox+signaling:+roles+in+cellular+stress+response,+stress+tolerance,+and+tissue+repair.">NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair.</a> Pharmacol. Rev. 63, 218-242 (2011).</li><li>Calafat, J., Kuijpers, T. W., 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+for+small+intracellular+vesicles+in+human+blood+phagocytes+containing+cytochrome+b558+and+the+adhesion+molecule+CD11b/CD18.">Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18.</a> Blood. 81, 3122-3129 (1993).</li><li>Johansson, A., Jesaitis, A. 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=Different+subcellular+localization+of+cytochrome+b+and+the+dormant+NADPH-oxidase+in+neutrophils+and+macrophages:+effect+on+the+production+of+reactive+oxygen+species+during+phagocytosis.">Different subcellular localization of cytochrome b and the dormant NADPH-oxidase in neutrophils and macrophages: effect on the production of reactive oxygen species during phagocytosis.</a> Cell. Immunol. 161, 61-71 (1995).</li><li>Kumar, A. P., Piedrafita, F. 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=Peroxisome+proliferator-activated+receptor+gamma+ligands+regulate+myeloperoxidase+expression+in+macrophages+by+an+estrogen-dependent+mechanism+involving+the+-463GA+promoter+polymorphism.">Peroxisome proliferator-activated receptor gamma ligands regulate myeloperoxidase expression in macrophages by an estrogen-dependent mechanism involving the -463GA promoter polymorphism.</a> J. Biol. Chem. 279, 8300-8315 (2004).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+inflammatory+phagocytes.">In vivo imaging of inflammatory phagocytes.</a> Chem Biol. 19, 1199-1209 (2012).</li><li>Gross, S., Gammon, S. 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=Bioluminescence+imaging+of+myeloperoxidase+activity+in+vivo.">Bioluminescence imaging of myeloperoxidase activity in vivo.</a> Nat. Med. 15, 455-461 (2009).</li><li>Kielland, A., Blom, 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=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radic. Biol. Med. 47, 760-766 (2009).</li><li>Zhou, J., Tsai, Y. 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=Noninvasive+assessment+of+localized+inflammatory+responses.">Noninvasive assessment of localized inflammatory responses.</a> Free Radic. Biol. Med. 52, 218-226 (2012).</li><li>Karlsson, A., Nixon, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phorbol+myristate+acetate+induces+neutrophil+NADPH-oxidase+activity+by+two+separate+signal+transduction+pathways:+dependent+or+independent+of+phosphatidylinositol+3-kinase.">Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.</a> J. Leukoc. Biol. 67, 396-404 (2000).</li><li>Taylor, R. G., McCall, C. E., 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=Histopathologic+features+of+phorbol+myristate+acetate-induced+lung+injury.">Histopathologic features of phorbol myristate acetate-induced lung injury.</a> Lab Invest. 52, 61-70 (1985).</li><li>Fukaya, S., Matsui, 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=Overexpression+of+TNF-alpha-converting+enzyme+in+fibroblasts+augments+dermal+fibrosis+after+inflammation.">Overexpression of TNF-alpha-converting enzyme in fibroblasts augments dermal fibrosis after inflammation.</a> Lab Invest. 93, 72-80 (2013).</li><li>Lu, Y. C., Yeh, W. 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=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42, 145-151 (2008).</li><li>Aitken, R. J., Buckingham, D. W., 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=Reactive+oxygen+species+and+human+spermatozoa:+analysis+of+the+cellular+mechanisms+involved+in+luminol-+and+lucigenin-dependent+chemiluminescence.">Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.</a> J. Cell. Physiol. 151, 466-477 (1992).</li><li>Allen, R. C., Loose, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+activation+of+a+luminol-dependent+chemiluminescence+in+rabbit+alveolar+and+peritoneal+macrophages.">Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages.</a> Biochem. Biophys. Res. Commun. 69, 245-252 (1976).</li><li>Caldefie-Chezet, F., Walrand, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+neutrophil+reactive+oxygen+species+production+measured+by+luminol+and+lucigenin+chemiluminescence+intra+or+extracellular?+Comparison+with+DCFH-DA+flow+cytometry+and+cytochrome+c+reduction.">Is the neutrophil reactive oxygen species production measured by luminol and lucigenin chemiluminescence intra or extracellular? Comparison with DCFH-DA flow cytometry and cytochrome c reduction.</a> Clin. Chim. Acta. 319, 9-17 (2002).</li><li>Dahlgren, C., Aniansson, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+formylmethionyl-leucyl-phenylalanine-induced+luminol-+and+lucigenin-dependent+chemiluminescence+in+human+neutrophils.">Pattern of formylmethionyl-leucyl-phenylalanine-induced luminol- and lucigenin-dependent chemiluminescence in human neutrophils.</a> Infect. Immun. 47, 326-328 (1985).</li><li>Dahlgren, C., Karlsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+burst+in+human+neutrophils.">Respiratory burst in human neutrophils.</a> J. Immunol. Methods. 232, 3-14 (1999).</li><li>Aerts, C., Wallaert, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+superoxide+anion+by+alveolar+macrophages+in+pulmonary+sarcoidosis.">Release of superoxide anion by alveolar macrophages in pulmonary sarcoidosis.</a> Ann. N.Y. Acad. Sci. 465, 193-200 (1986).</li><li>Zhang, N., Francis, K. P., 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=Enhanced+detection+of+myeloperoxidase+activity+in+deep+tissues+through+luminescent+excitation+of+near-infrared+nanoparticles.">Enhanced detection of myeloperoxidase activity in deep tissues through luminescent excitation of near-infrared nanoparticles.</a> Nat Med. , (2013).</li></ol>

저자는이 연구에서 관심 없음 충돌 선언하지 않습니다.

이 보고서에서, 우리는 살아있는 동물에서 염증의 비 침습 이미징을위한 생물 발광 방법을 보여줍니다. 두 개의 발광 기판, 루미놀 및 lucigenin을 활용하는 방법은 염증의 다른 단계를 구분할 수 있습니다. lucigenin의 생물 발광은 만성 단계에서 대식 세포에 의해 매개되는 반면 루미놀 생물 발광은 염증의 급성 단계에서 호중구와 연결되어 있습니다. 상대적으로 작은 (MW = 177.16 g / 몰)과 전기적으로 ?...

염증은 치유 2 상처, 당뇨병 3, 퇴행성 암 4, 심혈관 5, 6,자가 면역 질환 7 미생물 감염 1 등 인간 질병의 다양한 참여 규제가 엄격한 생물 학적 반응이다. 조직의 염증은 병원균 정리, 조직 복구, 질병 해상도를 달성하기 위해 다양한 면역 세포의 적절한 조정이 필요합니다. 호중구와 대식 세포 조직 염증의 주요 면역 매개체이다. 염증의 급성 단계에서는 백혈구는 각종 유해 자극 및 조직 손상 8 번째 조치입니다. 호중구는 빠르게 세포가 항 미생물 과립 및 식균 작용을 출시하여 미생물을 침해 비활성화 곳, 순환 부상의 사이트에 extravasate. 식균 작용하는 동안, 호중구 세포가 superox의 높은 수준을 생산하는 내 phagosomes를에 미생물을 침해 침몰IDE (O 2 · -). Phagosomal 과산화물은 많은 스트림 반응성 산소 종 (ROS)의 기본 소스입니다. 예를 들어, 초과 산화물은 자연 dismutation 또는 옥사이드 디스 뮤 타제 (SOD) 9, 10에 의해 과산화수소 (H 2 O 2)에 dismutated 할 수 있습니다. 호중구에서 myeloperoxidase는 (MPO) 더 항균 차아 염소산 (유리 HOCl) 11 과산화수소로 변환합니다. 염증 반응이 지속적으로 순환 단핵구은 점차 부상의 사이트에 마이그레이션하고 그 식세포 기능을 비활성화 병원균과 세포 파편을 제거하는 데 도움이 성숙한 대식 세포 2,로 구분. 또한, 염증의 이후 단계에서 중요한 규칙으로, 대식 세포는 항 염증성 사이토 카인 12 생산에 의해 그리고 낮은 레벨 9에서 세포 ROS를 생성하여 조직의 복구를 촉진합니다. 이 이후 단계에서 생성 된 ROS는 조직 리모델링, 새로운 혈관 형성 및 reepithelia을 조절lization 13. 식세포 NADPH 산화 효소 (Phox)는 호중구와 대 식세포 9 모두에서 초과 산화물의 생산의 주요 원천입니다. Phox 누구 조립 단단히 9 규제 멀티 소단위 복잡합니다. holoenzyme 여러 세포질 규제 소단위 (P67 phox, P47 phox, P40 phox 및 RAC)와 막 바인딩 heterodimer 시토크롬 B 558 (소단위 CYBA 및 CYBB으로 구성)이 포함되어 있습니다. 시토크롬 B 558 CYBB 소단위는 (또한 P91 phox 및 NOX2라고도 함) 차 산화 환원 연쇄 반응 9을 수행하는 내 반응이 핵심입니다. 흥미롭게도, 그 조립 사이트는 호​​중구와 대식 세포 사이에 차이가 있습니다. 휴식 호중구에서 시토크롬 B 558 세포 내 저장 과립 (14)의 막에 주로 존재한다. 식균 작용하는 동안, 호중구 H 조립MPO 활동의 높은 수준도 존재 phagosomes를 9 일에 oloenzymes. 호중구 Phox 빠르게 산소를 소비하고 ROS 생산하여 살균 능력을 발휘하는 현상은 호흡 버스트 11라고합니다. 대조적으로, 대식 세포는 MPO 표현의 낮은 수준을 가지고 있고 B 558은 주로 플라즈마 막 15, 16에서 발견된다 시토크롬. 대식 세포가 규제 기능 15 이하 과산화물을 생성하는 동안 따라서 호중구, 항균성 활동을 초과 산화물의 높은 수준을 생산하고 있습니다. 염증이 생체 과정에서 복잡한이기 때문에, 염증의 다른 단계에 대한 구체적인 비 침습 이미징 방법은 질병 모델의 정량적 경도 평가를 허용합니다. 기계론의 연구를 사용하여, 우리는 이전에 두 화학 발광 에이전트의 사용 루미놀 (5 - 아미노 -2,3 - 디 하이드로 -1,4 - phthalazinedione)와 lucigenin (BIS-N-methylacridinium 질산)를 증명하고있다각각 17 염증의 급성 및 후기 (만성) 단계의 비 침습 이미징을위한. lucigenin의 생물 발광은 후반 단계 또는 만성 염증 17과 관련있는 대식 세포의 활동을 평가하는 데 사용할 수있는 반면, 루미놀은 염증 18-20의 급성 단계에서 호중구 MPO 활동을 시각화 할 수 있습니다. 이 논문에서, 우리는이 이미징 기술을 보여주기 위해 두 가지 실험 염증 모델 (SC PMA 및 SC LPS)를 사용.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vivo imaging method to distinguish acute and chronic inflammation

염증은 많은 인간의 질병의 근본적인 측면이다. 이 비디오 보고서에서, 우리는 마우스 모델에서 급성 및 만성 염증을 구별 비 침습적 생물 발광 영상 기술을 보여줍니다. 조직 손상이나 병원체 침공, 호중구는 급성 염증 반응을 중재에 중요한 역할을 방어의 첫 번째 라인이다. 염증 반응이 진행되면서, 순환 단핵구은 점차 부상의 사이트에 마이그레이션 및 만성 염증을 중재 및 조직 파편을 제거하고 항 염증 사이토 카인을 생산하여 조직 복구를 촉진 성숙한 식세포로 분화. 루미놀의 복강 내 주입 (5 - 아미노 -2,3 - 디 하이드로 -1,4 - phthalazinedione, 나트륨 염)은 주로 조직 침투 호중구에 의해 중재 급성 염증의 검출을 할 수 있습니다. 루미놀은 특히 myeloperoxidase (M에서 생물 발광의 결과 때문에 호중구의 phagosomes를 내 생성 된 과산화물과 반응PO) 매개 반응. Lucigenin (BIS-N-methylacridinium 질산)는 생물 발광을 생성하기 위해 초과 산화물과 반응. 그러나 lucigenin의 생물 발광은 MPO 독립적이며 전적으로 만성 염증시 대 식세포의 식세포 NADPH 산화 효소 (Phox)에 의존합니다. 함께 루미놀과 lucigenin 비 침습 시각화 및 급성 및 만성 염증 단계에 걸쳐 서로 다른 식세포 인구의 경도 평가를 할 수 있습니다. 인간의 질병의 다양한 염증의 중요한 역할을 감안할 때, 우리는이 비 침습 영상 방법은 병적 인 조건의 다양한 호중구와 대 식세포의 차동 역할을 조사 할 수 있습니다 믿습니다.

우리는 실험 염증 모델에서 급성 및 만성 염증을 평가하는 종 생물 발광 영상을 시행 하였다. Phorbol 12 미리 스틴 산 13 - 아세테이트 (PMA)는 수퍼 옥사이드 음이온 생산을위한 Phox을 활성화하고 21 강렬한 급성 염증 반응을 유발 강력한 단백질 키나제 C (PKC) 작용제이다. NCR 누드 마우스에있는 PMA의 50 MG의 SC 주입 주사 부위 18 일 22시 빠른 피부 자극 및 급성 염증, 23를 일으켰?...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

우리는 염증 단계를 구별하기위한 비 침습 이미징 방법을 설명합니다. 루미놀의 전신 배달 호중구의 MPO 활동에 의존 급성 염증의 영역을 보여준다. 대조적으로, lucigenin의 주입은 대 식세포에서 Phox 활동에 의존 만성 염증으로 시각화 할 수 있습니다.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

in-vivo-imaging-method-to-distinguish-acute-and-chronic-inflammation

Research

JoVE Journal

Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

20.1K 조회수.  Harvard Medical School. 우리는 염증 단계를 구별하기위한 비 침습 이미징 방법을 설명합니다. 루미놀의 전신 배달 호중구의 MPO 활동에 의존 급성 염증의 영역을 보여준다. 대조적으로, lucigenin의 주입은 대 식세포에서 Phox 활동에 의존 만성 염증으로 시각화 할 수 있습니다.

비디오: In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

백혈구 호밍 및 마이 그 레이션의 비침습 영상 생체내에</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

연구

면역학 및 감염병학

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

로컬 및 전신 사이트에서 병원체에 의한 만성 염증을위한 마우스 모델

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

마우스 비장에서 불변 자연 살해 T 세포를 분리하고 확장을위한 최적화 방법

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

비 침습적<em&gt; In Vivo</em&gt; 활성화 가능한 형광 이미징 에이전트를 사용한 염증성 MMP 활성의 형광 광학 이미징

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

라이트 사이드에와 서: Vivo에서 당뇨병 털 Murine 모델에 만성 상처에서 녹 농 균 Biofilm 감염의 모니터링

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK과 만성 활성 엡 스타인-바 바이러스 감염을 가진 환자에서 T 셀 라인을 설정 하는 효율적이 고 간단한 방법

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

초파리 번데기 내에서 염증 성 세포 역학의 장기 Vivo에서 추적

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...