이 연구는 알레르기 및 감염성 질병, 건강의 국립 연구소의 국립 연구소의 교내 연구 부문에 의해 지원되었다. 우리는이 연구에서 사용 H57의 scFv으로 우리를 제공하는 요하네스 Huppa과 마크 데이비스에게 감사합니다. RV가이 기술의 개발시 유용한 논의 Keir 노이만 감사드립니다.

<ol>
	<li>Call, M. E., Wucherpfennig, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+receptor:+critical+role+of+the+membrane+environment+in+receptor+assembly+and+function.">The T cell receptor: critical role of the membrane environment in receptor assembly and function.</a> Annu. Rev. Immunol. 23, 101-125 (2005).</li><li>Smith-Garvin, J. E., Koretzky, G. A., Jordan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S.T+cell+activation.">S.T cell activation.</a> Annu. Rev. Immunol. 27, 591-619 (2009).</li><li>Bezbradica, J. S., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+cytokine+and+heterologous+receptor+signaling+pathways.">Integration of cytokine and heterologous receptor signaling pathways.</a> Nat. Immunol. 10, 333-339 (2009).</li><li>Fraser, I. D., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+the+network:+signaling+cross-talk+in+hematopoietic+cells.">Navigating the network: signaling cross-talk in hematopoietic cells.</a> Nat. Immunol. 10, 327-331 (2009).</li><li>Dustin, M. L., Starr, T., Varma, R., Thomas, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+planar+bilayers+for+study+of+the+immunological+synapse.">Supported planar bilayers for study of the immunological synapse.</a> Curr. Protoc. Immunol. Chapter 18, 13-13 (2007).</li><li>Vardhana, S., Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+Planar+Bilayers+for+the+Formation+of+Study+of+Immunological+Synapses+and+Kinapse.">Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse.</a> J. Vis. Exp. (19), e947-e947 (2008).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster.">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25, 117-127 (2006).</li><li>Yokosuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat. Immunol. 6, 1253-1262 (2005).</li><li>Huppa, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463, 963-967 (2010).</li><li>Tolar, P., Pierce, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conformation-induced+oligomerization+model+for+B+cell+receptor+microclustering+and+signaling.">A conformation-induced oligomerization model for B cell receptor microclustering and signaling.</a> Curr. Top. Microbiol. Immunol. 340, 155-169 (2010).</li><li>Treanor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+skeleton+controls+diffusion+dynamics+and+signaling+through+the+B+cell+receptor.">The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.</a> Immunity. 32, 187-199 (2010).</li><li>Treanor, B., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insight+into+lymphocyte+activation+through+quantitative+imaging+and+theoretical+modelling.">Mechanistic insight into lymphocyte activation through quantitative imaging and theoretical modelling.</a> Curr. Opin. Immunol. 19, 476-483 (2007).</li><li>Sylvain, N. R., Nguyen, K., Bunnell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vav1-mediated+scaffolding+interactions+stabilize+SLP-76+microclusters+and+contribute+to+antigen-dependent+T+cell+responses.">Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses.</a> Sci. Signal. 4, ra14-ra14 (2011).</li><li>Purbhoo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+subsynaptic+vesicles+and+surface+microclusters+at+the+immunological+synapse.">Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.</a> Sci. Signal. 3, ra36-ra36 (2010).</li><li>Lasserre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezrin+tunes+T-cell+activation+by+controlling+Dlg1+and+microtubule+positioning+at+the+immunological+synapse.">Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.</a> EMBO. J. 29, 2301-2314 (2010).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7:+Total+internal+reflection+fluorescence+microscopy.">Chapter 7: Total internal reflection fluorescence microscopy.</a> Methods Cell. Biol. 89, 169-221 (2008).</li><li>Williamson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existing+clusters+of+the+adaptor+Lat+do+not+participate+in+early+T+cell+signaling+events.">Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events.</a> Nat. Immunol. 12, 655-662 (2011).</li></ol>

이 실험에 사용된 모든 동물들은 무균 환경에서 유지되었으며, 실험은 보건 동물 관리 및 사용위원회의 국립 연구소에 의해 승인되었다.

우리는 여기서 TIRFM과 인조 APCs과 같은 유리를 지원 지질 bilayers를 사용하여 항원에 특정한 기본 마우스 T 세포에 신호를 공부하는 시스템을 설명합니다. 기술은 성공적으로 이들 세포에 GFP 태그가 단백질을 표현에 의존합니다. T 세포를 Transfecting 것은 언제나 어려운 작업입니다. 일반적으로 retroviruses 또는 lentiviruses을 사용하여 electroporation이나 유전자 전달이 사용됩니다. 한 기술은 다른여 우수하지 않으며 모두 자신의 한계와 장점이 있습니다. 1) 이는 retroviruses 아니지만 lentiviruses 필요로 세포가 활발히 분열하는 것이 필요하지 않습니다, 그리고 2) 표현 수준이 세포 이미징 전에 incubated되는 시간을 변화하여 제어할 수 있습니다 : 우리는 electroporation은 다음과 같은 장점이있다 찾습니다. 가장 큰 단점은 우리가 매우 어려운 일차 세포의 크기가 큰 단백질을 표현하는 것입니다. 우리는 electroporation 이후 우리에게 아주 좋은 세포 생존 능력을 제공 방법을 제시했습니다. 결과적으로 그것은 applicab입니다siRNA의 중재 유전자 입을를 달성하기 위하여 그것을 사용 르. 우리는 또한 여기서 chromatically 수정하고 다른 파장에 대해 별도의 정렬이 필요없는 맞춤형 두 채널 동시 수집 TIRF 현미경을 설명합니다. 이러한 기능은 있지만 상업적으로 사용할 수 있습니다. 저희 시스템은 TIRF 현미경 분야의 전문가에 의해 출판 및 상업 대안보다 저렴의 원리에 근거한다. 우리 디자인의 한 가지 비판은 우리가 다른 여기 파장에 대한 발생률의 동일한 각도를 사용하고있다는 것입니다. 이것은 다른 여기 파장에 대해 다른 TIRF의 침투 깊이가 나타날 수 있습니다. 우리는 지극히 미미한 파도가 기하 급수적으로 부패 분야이며 TIRF 필드의 깊이가 파장의 선형 함수이기 때문에 이러한 효과가 작기 생각합니다. 유리 표면에 가까운 Fluorophores는 두 파장 사이의 레이저 농도에는 차이가 발생할하지 않습니다. PE 근처 fluorophores위한netration 깊이는 강도 차이는 두 파장 488 및 561 nm의를위한 두 파장 488 및 640 nm의 및 1.15 배위한 1.3 배 될 것입니다. 동일한 시스템 TIRF 조명을 사용하게 수퍼 해상도 기술과 함께 사용될 수 있습니다. 우리는 또한 사용자 정의 내장입니다 두 카메라의 시스템을 설명했습니다. TIRF 시스템과 마찬가지로 유사한 apparatuses 또한 상업적으로 사용할 수 있습니다. Google 시스템은 파장의 서로 다른 조합과의 사용할 수 있도록 필터와 dichroics을 변경하는 유연성을 제공합니다. 시스템의 단점은 두 이미지를 정렬하는데 필요한 회전 한 정도입니다. 이 문제는 압전 구동과 정렬의 회전 정도를 제공하고 있습니다 카메라 스탠드를 사용하여 해결할 수 있습니다. 우리는 또한 성공적으로 카메라는 다른 서로 다른 픽셀 크기를 가지고있는이 현미경에 두 개의 카메라 시스템을 구축했습니다. 하나 하나의 채널에서 민감도와 대형 다이 필요한 경우이 같은 시스템이 있으면 좋을 텐데또 다른 namic 범위는, 둘 다 같은 카메라에 거의 사용할 수 없습니다. 우리는 16 μm의의 픽셀 크기를 가지고 Photometrics 양의-EM 카메라와 12.9 μm의의 효과적인 픽셀 크기를 제공 2x2 binning에 Photometrics HQ-2 카메라를 사용했습니다. 180mm 초점 거리 튜브 렌즈는 양의-EM에 사용되었으며, 145mm 초점 거리 튜브 렌즈는 HQ-2 카메라 사용되었다. HQ-2 Metamorph 소프트웨어와 양의-EM은 외부 트리거 모드에서 별​​도의 컴퓨터에 마이크로 매니저 소프트웨어를 통해 제어습니다을 사용하여 통제되었다. 외부 트리거는 Metamorph의 구성 조명 설정에서 추가로 셔터로 구현된 측정 컴퓨팅 DAC 보드의 디지털 출력을 사용하여 양의-EM 카메라에 제공되었다. 튜브 렌즈의 초점 길이의 비율은 유효 픽셀 크기의 비율과 일치합니다. 대표 정렬은 그림 5에 표시됩니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 마우스 T 세포에 대한 Amaxa Nucleofector 키트 </td><td> Lonza 주식 회사 </td><td> VPA-1006 </td><td></td></tr><tr><td> PureLink HiPure 플라스미드 Midiprep 키트 </td><td> 생명 기술 </td><td> K2100-05 </td><td> 내독소 무료 디엔에이 preps위한 </td></tr><tr><td> Amaxa Nucleofector 장치 </td><td> Lonza 주식 회사 </td><td> AAD-1001 </td><td></td></tr><tr><td> 재조합 Murine IL-2 </td><td> Peprotech 주식 회사 </td><td> 212-12 </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent Microspheres 0.20μm </td><td> Polysciences 주식 회사 </td><td> 24050-5 </td><td></td></tr><tr><td> 실험실 - 테크 II 8-잘 chambered coverglass </td><td> 써모 과학, Nunc </td><td> 155,409 </td><td></td></tr></tbody></table>

a tirf microscopy technique for real-time, simultaneous imaging of the tcr and its associated signaling proteins

그것이 주요 Histocompatibility 컴플렉스 (pMHC) 항원 제시 세포 (APCs)의 표면에 표현 단백질에 의해 구속 antigenic의 펩타이드 조각으로 종사하는 경우 신호는 T 세포 수용체 (TCR)를 통해 시작됩니다. TCR 복합은 리간드 바인딩 TCRαβ의 heterodimer로 구성되어 있으며 그 동료 인 경우에는 3 번 CD dimers (εδ 및 εγ heterodimers 및 ζζ homodimer) 1 비 covalently. 수용체의 교전시 인 경우에는 3 번 CD의 ζ 체인은 Lck, SRC 가족 키나제에 의해 phosphorylated된다. 이것은 그때 Lck 의해 phosphorylated 및 활성화 Syk 가족 키나제, Zap70의 채용으로 연결됩니다. 그 후, Zap70 다른 신호 분자 2의 다수를 포함하는 근위 신호 단지의 형성을 시작, 어댑터 단백질 LAT와 SLP76을 phosphorylates. 칼슘과 RAS 종속 tra이 복잡한 결국 결과의 형성nscription 요소 활성화 및 T 세포 분화 2 야기할 유전자 발현 프로그램의 복잡한 일련의 논리상 필연의 개시. TCR 신호 (및 분화의 결과 상태)는 항원 힘 및 누화 co-stimulatory/co-inhibitory와 케모카인 및 3-4 시토킨 수용체를 포함한 기타 여러 요인에 의해 변조된 있습니다. 다양한 자극 조건에서 근위 신호 복합 공간과 시간적 조직을 공부한다는 것은 따라서, TCR 신호 경로뿐만 아니라 다른 신호 전달 경로에 의해 그 규정을 이해하는 열쇠입니다. T 세포의 플라즈마 막에서 TCR에 의해 시작 신호를 공부하는 한 매우 유용한 모델 시스템은 이전에 설명한 5-6 유리 지원 지질 bilayers입니다. 그들은 인공 APCs으로 T 세포 자찬에 antigenic의 pMHC 단지, 접착 및 공동 stimulatory 분자를 제시 활용할 수 있습니다. 영상으로 T 세포와 상호 작용총 내부 반사 형광 현미경 (TIRFM)를 사용하여 지질 이중층, 우리는 7-8 유리와 세포 표면 사이의 공간을 100 nm의 범위까지 여기를 제한할 수 있습니다. 이것은 우리가 이미지에 주로 신호​​ 이벤트 플라즈마 막에서 발생하실 수 있습니다. 우리 이미징에서 TCR은 복합 단백질 신호의 채용에 관심이있다, 우리는 두 카메라 TIRF 이미징 시스템을 설명하는 어떤 점에서 H57 항체의 형광 팹으로 표시 TCR은 (단편 항원 결합) 조각 (하이 브리 도마 H57-597에서 정화 , ATCC, ATCC 번호 : GFP 태그가 TCRβ에 대한 구체적인 HB-218), 그리고 신호 전달 단백질은, 동시에 실시간으로 몇 군데있을 수 있습니다. 이 전략은 T 세포 모두와 TCR에서 발생하는 신호 이벤트의 높은 동적 특성으로 인해 필요합니다. 이 영상 양상 이미지 단일 리간드 9-11뿐만 아니라 활성화된 수용체에 분자를 신호 모집과 연구자를 허용하고있다12-16 원위치에서 생화학을 공부하는 훌륭한 시스템입니다.

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

따라서, 그것이 관여 단백질의 공간적 및 시간적 행위를 파악하는 것이 중요 신호 전달 이해하기 위해, 플라즈마 막 내에서 또는 세포 내 위치에 두 단백질의 compartmentalization 크게 신호 결과에 영향을 미칠 수있는 규제 메커니즘입니다. 우리는 여기서 T 세포의 신호 전달을 연구하는 TIRF 현미경 기반 시스템을 설명하지만, 광범위하게 적용됩니다.

실시간, TCR의 동시 이미징 및 관련 신호 전달 단백질에 대한 TIRF 현미경 기법

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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JoVE Journal

Immunology and Infection

23.7K 조회수.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 따라서, 그것이 관여 단백질의 공간적 및 시간적 행위를 파악하는 것이 중요 신호 전달 이해하기 위해, 플라즈마 막 내에서 또는 세포 내 위치에 두 단백질의 compartmentalization 크게 신호 결과에 영향을 미칠 수있는 규제 메커니즘입니다. 우리는 여기서 T 세포의 신호 전달을 연구하는 TIRF 현미경 기반 시스템을 설명하지만, 광범위하게 적용됩니다.

비디오: A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

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

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

마우스의 림프 Vasculature의 비 침습적 광학 이미징

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

골수 세포 역학의 실시간 영상<em&gt; APC<sup&gt; 최소 / +</sup</em&gt; 소장 종양 디스크 공 초점 현미경을 회전시켜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

분석<em&gt; 예 르시 니아의 enterocolitica</em숙주 세포 내로&gt; 이펙터 전좌 베타 - 락타 마제 이펙터의 용해 사용

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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 ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

확산의 분석 및 형광 현미경 검사 법에 의해 수용 체 막의 클러스터 크기에 대 한 프로토콜을 처리 하는 이미지

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...