GFP - β - 굴지 플라스미드는 넉넉한 박사 웨인 오어 병리의 LSUHSC - S 부서에 의해 제공되었고, 박사 베키 Worthylake, 약리학의 LSUHSC - NO학과 실험실에서 증폭되었다. 이 작품은 국립 보건원 (P20 RR - 018766)와 미국 심장 협회 (05835386N)에서 부여에 의해 지원되었다.

<ol>
	<li>Spindler, V., Schlegel, N., Waschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTPases+in+control+of+microvascular+permeability.">Role of GTPases in control of microvascular permeability.</a> Cardiovasc. Res. 87, 243-253 (2010).</li><li>Wojciak-Stothard, B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+and+the+regulation+of+endothelial+permeability.">Rho GTPases and the regulation of endothelial permeability.</a> Vascul. Pharmacol. 39, 187-199 (2002).</li><li>Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+pathways+in+enhanced+microvascular+permeability.">Signal transduction pathways in enhanced microvascular permeability.</a> Microcirculation. 7, 395-403 (2000).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+in+mechanosensitive+regulation+of+endothelial+barrier.">Small GTPases in mechanosensitive regulation of endothelial barrier.</a> Microvasc. Res. 77, 46-52 (2009).</li><li>Duran, W. N., Sanchez, F. A., Breslin, J. W., Tuma, R. F., Duran, W. N., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+Exchange+Function.">Microcirculatory Exchange Function.</a> Handbook of Physiology: Microcirculation. , 81-124 (2008).</li><li>Hu, Y. L., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+motion+of+paxillin+on+actin+filaments+in+living+endothelial+cells.">Dynamic motion of paxillin on actin filaments in living endothelial cells.</a> Biochem. Biophys. Res. Commun. 357, 871-876 (2007).</li><li>Borm, B., Requardt, R. P., Herzog, V., Kirfel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ruffles+in+cell+migration:+indicators+of+inefficient+lamellipodia+adhesion+and+compartments+of+actin+filament+reorganization.">Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.</a> Exp. Cell. Res. 302, 83-95 (2005).</li><li>Hotulainen, P., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+are+generated+by+two+distinct+actin+assembly+mechanisms+in+motile+cells.">Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.</a> J. Cell. 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J. 84, 2691-2699 (2003).</li><li>Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G., Verin, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Rho+GTPases+in+thrombin-induced+lung+vascular+endothelial+cells+barrier+dysfunction.">Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction.</a> Microvasc. Res. 67, 64-77 (2004).</li><li>van Nieuw Amerongen, G. P., van Delft, S., Vermeer, M. A., Collard, J. G., van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+RhoA+by+thrombin+in+endothelial+hyperpermeability:+role+of+Rho+kinase+and+protein+tyrosine+kinases.">Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.</a> Circ. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+techniques+to+study+mobility+and+molecular+dynamics+of+proteins+in+live+cells:+FRAP,+iFRAP,+and+FLIP.">Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP.</a> Live Cell Imaging: A Laboratory Manual. , 101-126 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

라이브 내피 세포에 GFP - 굴지의 영상은 염증성 자극에 대한 응답으로 굴지의 cytoskeleton의 역학에 대한 자세한 분석을 수 있습니다. 이 방법은 또한 전단 응력 11과 같은 물리적 세력에 대응 cytoskeleton의 리모델링을 보여주는 이전 연구 결과에 기초하여 유용할 수 있습니다. 또한,이 방법은 마이 그 레이션, 유사 분열, 세포 분기점 및 junctional 성숙의 형성, 그리고 장벽 기능의 유지 보수 등 다양한 내피 세포 활동에 굴지 cytoskeletal 역학의 공헌에 대한 자세한 평가를하실 수 있습니다. 표시된 데이터의 내피 굴지의 cytoskeleton의 동작은 트롬빈 치료 전후에 볼 수 있습니다. 모든 내피 세포의 가장자리를 따라 로컬 lamellipodia은 성형과 nonconfluent과 합류 모두 세포 monolayers에 시간이 지남에 따라 regressing 관찰되었다. 트롬빈과 치료는 간단히 lamellipodia 양식을 중단ation과 매상. 트롬빈은 트롬빈이 내피 세포의 12-14 굴지 스트레스 섬유 형성 및 증가 구심 긴장 개발을 일으키는 이전 리포트와 계약에, 세포가 약간 계약에 발생했습니다. 그러나, 이와 같은 라이브 세포 이미징 연구에서 스트레스 섬유의 출처는 현재 확인할 수 있습니다. HUVEC에서 스트레스 섬유의 대부분은 세포 주변에서 발생한 및 세포 8, 9를 마이 그 레이션에 가로 아크 섬유를 닮은. 고정 세포를 사용하여 이상이 방법의 또 다른 장점은 스트레스 섬유의 수를 실험 그룹간에 선택 바이어스를 제거, 트롬빈​​ 치료 전후 각 세포에 계량 수있다는 것입니다. 이 프로토콜을 통해 우리는 셀 가장자리와 굴지의 스트레스 섬유의 역동적인 움직임을 평가합니다. photobleaching 후 내피 세포와 같은 형광 복구와 같은 고급 기술에 굴지의 모노머의 역학을 이해 (FRAP) 또는 형광는 마이크로을 작은 반점하기복사 (FSM)는 16 일 15 적용할 수 있습니다. 또한, 때문에 microvascular 내피 세포는 microvascular 장벽 기능의 더 나은 모델을 효과적으로 논리적인 미래 방향을 나타냅니다 microvascular 내피 세포에 GFP - 굴지 표현 transfection 프로토콜의 최적화를 대표하는 수도 있습니다. 요약, GFP - 굴지 표현 라이브 내피 세포의 이미징은 내피 세포 굴지의 cytoskeleton은 자극에 다양한 종류의에 응답하는 방법을 결정하는 강력한 도구를 나타냅니다. 단단히 합류 내피 monolayers를 사용하는 연구는 굴지 풍부한 lamellipodia와 내피 장벽 기능에 가로 아크 스트레스 섬유와 같은 역동적인 구조의 역할을 결정하는 데 도움이됩니다. 또한, GFP - 굴지 또는 기타 subcellular 구조의 시각화 수 있도록 다른 융합 단백질을 표현하는 내피 세포의 라이브 세포 이미징은 신호 및 구조 메커니즘을 이해하는 데 필요한 자세한 spatiotemporal 정보를 제공합니다 것이 Determine 장벽의 무결성.

<table border="1"><tbody><tr><td colspan="4"> 1. 닮은 5 배 증권 </td></tr><tr><td> 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 나트륨 염화물 </td><td> EMD </td><td> SX0420 - 3 </td><td> 35g </td></tr><tr><td> 염화칼륨 </td><td> JT 베이커 </td><td> 3040 </td><td> 1.75 g </td></tr><tr><td> 염화칼슘 </td><td> 시그마 </td><td> C - 3881 </td><td> 1.47 g </td></tr><tr><td> 마그네슘 황산 </td><td> 시그마 </td><td> M - 9397 </td><td> 1.44 g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td colspan="4"> 4 autoclaved 병 및 상점으로 살균 필터 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2. 맙스 버퍼 </td></tr><tr><td> 화학 </td><td>회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 맙스 </td><td> 시그마 </td><td> M3183 </td><td> 125.6 g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td colspan="4"> 4 autoclaved 병 및 상점으로 살균 필터 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3. 알부민 생리 식염수 (APSS) </td></tr><tr><td> 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 닮은 주식 (5 배) </td><td> N / A </td><td> N / A </td><td> 200 ML </td></tr><tr><td> 맙스 버퍼 </td><td> N / A </td><td> N / A </td><td> 5 ML </td></tr><tr><td> 인산 나트륨 </td><td> 시그마 </td><td> S - 9638 </td><td> 0.168 g </td></tr><tr><td> 나트륨 Pyruvate &lt;/ TD&gt; <td> 시그마 </td><td> P5280 </td><td> 0.22 g </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) 나트륨 소금 </td><td> 시그마 </td><td> ED2SS </td><td> 0.0074 g </td></tr><tr><td> 포도당 </td><td> 시그마 </td><td> G7528 </td><td> 0.901 g </td></tr><tr><td> 알부민, 소의 </td><td> USB </td><td> 10,856 </td><td> 10g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td colspan="4"> 37 7.4로 산도를 조절 ° C, 4 autoclaved 병 및 저장에 다음 살균 필터 ° C. </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4. 0.9 %의 염분 </td></tr><tr><td> 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 나트륨 염화물 </td><td> EMD </td><td> SX0420 - 3 </td><td> 9g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td colspan="4"> 4 autoclaved 병 및 상점으로 살균 필터 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5. 1.5 % 다행 솔루션 </td></tr><tr><td> 돼지의 피부에서 젤라틴 </td><td> 시그마 </td><td> G2500 </td><td> 15g </td></tr><tr><td> 0.9 %의 염분 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td colspan="4"> 37에 대한 해결책을 따뜻하게 ° C 충분히 젤라틴 디졸브 수 있습니다. 동안 4 autoclaved 병 및 저장에 여전히 따뜻하고, 살균 필터 ° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

microvascular 내피는 유체와 solutes에 선택적으로 투과 장벽으로 중요한 역할을합니다. 내피 세포 사이의 접착제 분기점은 내피의 투과성을 조절하고, 많은 연구가 junctional 무결성 1-5 결정하기 위해 굴지의 cytoskeleton의 중요한 기여를 표명했습니다. 피질 굴지 벨트는 안정적인 분기점 1, 2, 4, 5의 유지에 중요한 것으로 생각됩니다. 반대로, 굴지의 스트레스 섬유 분기점 2-5 약화 내피 세포 내에서 구심력 장력을 생성하는 생각입니다. 대부분이 이론은 내피 세포가 내피 투과성을 증가하는 것으로 알려져 염증 중재자로 치료 후 세포를 고정하고 현미경 관찰을위한 F - 굴지 분류하는 연구를 기반으로하고 있습니다. 에서 수정된 세포의 이미지만을 스냅샷을 제공하기 때문에 그러나 이러한 연구는 굴지의 cytoskeleton의 역할이 매우 제한된 이해를 제공합니다굴지의 구조 5 역학에 대한 정보와 시간. 라이브 세포 이미징은 내피 장벽 무결성을 결정하는 메커니즘의 연구에 굴지의 cytoskeleton의 동적 특성의 결합을 허용합니다. 이 방법의 가장 큰 장점은 내피 세포에서 굴지의 구조에 대한 다양한 염증성 자극의 영향이 고정 표본을 관찰하는 경우에 발생할 수있는 잠재적인 편견을 제거하기 전에 살아있는 세포의 동일한 세트에서 치료 후 평가 될 수있다는 것이다. 인간 제대 정맥 내피 세포 (HUVEC)는 플라스미드 GFP - β - 굴지로 transfected 유리 coverslips에 합류하기 위해 성장하고 있습니다. 합류 HUVEC에서 GFP - 굴지의 시간 저속 이미지는 내피 장벽 무결성 시간 종속적인 변화를 이끌어내는 염증 중재자의 추가 전후 캡처됩니다. 이러한 연구는 endothelia에 기여하는 굴지의 cytoskeleton의 변화 유체 순서의 시각적 관찰을 활성화내 장벽 파괴 및 복원. 우리의 결과는 지속적으로 내피 세포의 지방, 굴지 풍부한 lamellipodia 형성과 매출을 보여줍니다. 굴지의 스트레스 섬유의 형성과 움직임도 볼 수 있습니다. 염증성 자극 치료 전후 지역 lamellipodia의 형성과 매상의 주파수 분석은 kymograph 분석하여 문서화 수 있습니다. 이러한 연구는 내피 장벽의 무결성의 유지를위한 중요한 이전에 정체 불명의 분자 메커니즘을 발견하는 데 사용할 수있는 내피 세포에있는 굴지의 cytoskeleton의 동적 특성에 대한 중요한 정보를 제공합니다.

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

GFP - 굴지 표현 라이브 내피 세포의 미세 영상은 cytoskeletal 구조에서 동적 변화 특성화 수 있습니다. 고정 표본을 사용하는 기술과는 달리,이 방법은 전에 동일한 세포에있는 굴지의 cytoskeleton의 시간적 변화에 대한 자세한 평가, 동안 다양한 물리적, 약리, 또는 염증성 자극 후를 제공합니다.

GFP - 굴지 표현 라이브 내피 세포 굴지의 Cytoskeleton 연구

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

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Unit 1

The Cytoskeleton I: Actin and Microfilaments

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27.1K 조회수.  Louisiana State University Health Sciences Center. GFP - 굴지 표현 라이브 내피 세포의 미세 영상은 cytoskeletal 구조에서 동적 변화 특성화 수 있습니다. 고정 표본을 사용하는 기술과는 달리,이 방법은 전에 동일한 세포에있는 굴지의 cytoskeleton의 시간적 변화에 대한 자세한 평가, 동안 다양한 물리적, 약리, 또는 염증성 자극 후를 제공합니다.

비디오: Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

세포 운동성 및 Zebrafish 태아의 개별 뉴런과 신경 크레스트 세포의 굴지의 Cytoskeleton의 라이브 영상

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

에 대한 내피 세포의 관 형성의 분석<em&gt; 체외</em&gt; 혈관 신생 연구

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

제브라 피쉬 배아에서 산화 스트레스의 분석

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...