우리는 안티 Giantin 항체, M. Labouesse 랩으로 우리를 제공하기위한 L. Brino (IGBMC 높은 콘텐츠 상영 시설하여 Illkirch, 프랑스)을 인정합니다. C.에 대한 간스 (IGBMC)와 B Séraphin 연구소. 효모 (IGBMC), 분열 효모 세포에 대한 형광 HeLa 세포 (MPI-CBG, 드레스덴), J. 모슬리 (다트머스 의과 대학)와 JQ 우 (오하이오 주립 대학)에 대한 E. Paluch 및 A. 하이 먼 신진에 대한; A. Hoël과 실험 도움 F. Evenou, 기술 지원을위한 C. 릭 (IBMC, 스트라스부르, 프랑스), 및 미세에 도움 JC Jeannot (펨토 - 일, 프랑스). 이 작품은 CNRS, 스트라스부르 대학, Conectus에서 기금에 의해 지원되었다, 라 파운데이션 부문은 라 공들인 MEDICALE와 스트라스부르의 CI-FRC를 붓는다.

<ol>
	<li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+Cell-Matrix+Adhesions+to+the+Third+Dimension.">Taking Cell-Matrix Adhesions to the Third Dimension.</a> Science. 294, 1708-1712 (2001).</li><li>Azioune, A., Storch, M., Bornens, M., Thery, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9, 1640-1642 (2009).</li><li>Mandal, K., Balland, M., Bureau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+Micropatterned+Substrates+for+Single+Cell+Studies.">Thermoresponsive Micropatterned Substrates for Single Cell Studies.</a> PLoS ONE. 7, e37548 (2012).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat Rev Mol Cell Biol. 8, 839-845 (2007).</li><li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension:+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues.</a> J Cell Sci. , (2012).</li><li>Ghibaudo, M., Di Meglio, J. M., Hersen, P., Ladoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cell+spreading+within+3D-micropatterned+environments.">Mechanics of cell spreading within 3D-micropatterned environments.</a> Lab Chip. 11, 805-812 (2010).</li><li>Greiner, A. M., Richter, B., Bastmeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-Engineered+3D+Scaffolds+for+Cell+Culture+Studies.">Micro-Engineered 3D Scaffolds for Cell Culture Studies.</a> Macromol Biosci. 12, 1301-1314 (2012).</li><li>Khetan, 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=Degradation-mediated+cellular+traction+directs+stem+cell+fate+in+covalently+crosslinked+three-dimensional+hydrogels.">Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels.</a> Nat Mater. 12, 458-465 (2013).</li><li>Legant, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tractions+exerted+by+cells+in+three-dimensional+matrices.">Measurement of mechanical tractions exerted by cells in three-dimensional matrices.</a> Nat Meth. 7, 969-971 (2010).</li><li>Riveline, D., Buguin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Devices and methods for observing the cell division. , (2010).</li><li>Ochsner, 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=Micro-well+arrays+for+3D+shape+control+and+high+resolution+analysis+of+single+cells.">Micro-well arrays for 3D shape control and high resolution analysis of single cells.</a> Lab Chip. 7, 1074-1077 (2007).</li><li>Riveline, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Devices and methods for observing cells with cell wall or invertebrate embryos with oblong eggshell . , (2012).</li><li>Riveline, D., Wollrab, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Devices and methods for observing eukaryotic cells without cell wall. , (2012).</li><li>Zanella, F., Lorens, J. B., Link, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening:+seeing+is+believing.">High content screening: seeing is believing.</a> Trends Biotech. 28, 237-245 (2010).</li><li>Wolfe, D., Qin, D., Whitesides, G., Hughes, M. P., Hoettges, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineering+in+Biotechnology,+Ch+3.">Microengineering in Biotechnology, Ch 3.</a> Methods in Molecular Biology. 583, 81-107 (2010).</li><li>Mehling, M., Tay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+cell+culture.">Microfluidic cell culture.</a> Curr Op Biotech. 25, 95-102 (2014).</li><li>Wollrab, V., Thiagarajan, R., Wald, A., Kruse, K., Riveline, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Still+and+rotating+myosin+clusters+determine+cytokinetic+ring+constriction.">Still and rotating myosin clusters determine cytokinetic ring constriction.</a> Nat Commun. 7, 11860-11869 (2016).</li><li>Yao, X., 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=Functional+analysis+of+single+cells+identifies+a+rare+subset+of+circulating+tumor+cells+with+malignant+traits.">Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits.</a> Integr Biol. 6, 388-398 (2014).</li><li>Eberwine, J., Sul, J. Y., Bartfai, T., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+single-cell+sequencing.">The promise of single-cell sequencing.</a> Nat Meth. 11, 25-27 (2014).</li><li>Allen, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cell-free+extracts+of+Xenopus+eggs+and+demembranated+sperm+chromatin+for+the+assembly+and+isolation+of+in+vitro-formed+nuclei+for+Western+blotting+and+scanning+electron+microscopy+(SEM).">Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).</a> Nat. Protocols. 2, 1173-1179 (2007).</li><li>Robbins, E., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotically+Synchronized+Mammalian+Cells:+a+Simple+Method+for+Obtaining+Large+Populations.">Mitotically Synchronized Mammalian Cells: a Simple Method for Obtaining Large Populations.</a> Science. 144, (1964).</li><li>Whitfield, M. L., 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=Stem-loop+binding+protein,+the+protein+that+binds+the+3&amp;#34;+end+of+histone+mRNA,+is+cell+cycle+regulated+by+both+translational+and+posttranslational+mechanisms.">Stem-loop binding protein, the protein that binds the 3&amp;#34; end of histone mRNA, is cell cycle regulated by both translational and posttranslational mechanisms.</a> Molecular and Cellular Biology. 20, (2000).</li><li>Straight, A. F., 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=Dissecting+Temporal+and+Spatial+Control+of+Cytokinesis+with+a+Myosin+II+Inhibitor.">Dissecting Temporal and Spatial Control of Cytokinesis with a Myosin II Inhibitor.</a> Science. 299, 1743-1747 (2003).</li><li>Tang, J., Erikson, R. L., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoint+kinase+1+(Chk1)+is+required+for+mitotic+progression+through+negative+regulation+of+polo-like+kinase+1+(Plk1).">Checkpoint kinase 1 (Chk1) is required for mitotic progression through negative regulation of polo-like kinase 1 (Plk1).</a> Proc Natl Acad Sci. 103, 11964-11969 (2006).</li><li>Zahreddine, H., Zhang, H., Diogon, M., Nagamatsu, Y., Labouesse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRT-1/Calreticulin+and+the+E3+Ligase+EEL-1/HUWE1+Control+Hemidesmosome+Maturation+in+C.+elegans+Development.">CRT-1/Calreticulin and the E3 Ligase EEL-1/HUWE1 Control Hemidesmosome Maturation in C. elegans Development.</a> Curr Biol. 20, 322-327 (2010).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> JoVE. , e4019 (2012).</li><li>Saha, S., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin-related+protein+Mid1p+coordinates+the+assembly+of+the+cytokinetic+contractile+ring+in+fission+yeast.">Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast.</a> Mol Biol Cell. 23, 3982-3992 (2012).</li><li>Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Drug+Efficacy+and+Toxicology+in+Three+Dimensions:+Using+Synthetic+Extracellular+Matrices+in+Drug+Discovery.">Evaluating Drug Efficacy and Toxicology in Three Dimensions: Using Synthetic Extracellular Matrices in Drug Discovery.</a> Acc Chem Res. 41, 139-148 (2007).</li><li>Futamura, 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=Morphobase,+an+Encyclopedic+Cell+Morphology+Database,+and+Its+Use+for+Drug+Target+Identification.">Morphobase, an Encyclopedic Cell Morphology Database, and Its Use for Drug Target Identification.</a> Chem Biol. 19, 1620-1630 (2012).</li></ol>

우리는 공개 할게 없다.

복제 성형은 &#39;eggcups&#39;를 제조하기 위해 사용 하였다. 제조 공정은 클린 룸을 필요로하지 않는다; 연습이 필요할 수 있지만 그것은 쉽고 간단합니다. 특히, PDMS 스탬프을 떼면 고품질 &#39;eggcups&#39;의 넓은 영역을 생성하기 위해 가장 중요한 단계이다. 이 때문에, 특별한주의가이 단계에서 수행되어야한다. 이 단계를 반복하여 실패한 경우, 실란 화 플라즈마 결합 전에 플라즈마 클리너 파라미터...

시험 관내 세포 - 기반 검정에서의 전류 (2D) 이차원이다. 이 구성은 포유 동물 세포에 대한 자연 아니므 한 생리 관련이 없다; 세포 모양, 크기 및 이기종 표현형의 다양성을 보여줍니다. 같은 비행기 및 세포 소기관의 극단적 인 표현형 (특히 스트레스 섬유) 내에서 무질서한 분포로, 심사 응용 프로그램에 적용 할 때 그들은 추가 심각한 한계를 제시한다. 이는 높은 예산이 매년 소비되는 약물 검사에 대한 임상 시험에서 특히 중요하다. 때문에 약물 검사의 초기 단계에서 인공 2D 문화 조건의 동물 모델에 적용 할 때이 약의 대부분은 비록 실패합니다. 또한,이 방법을 이용하여 특정 세포 소기관 적절 통상 관찰의 평면에 수직 한 평면에서 진화 구조 같은 세포 분열 동안 cytokinetic 액토 마이 오신 고리로서 가시화 될 수 없다. 약간새로운 2 차원 분석은 세포 골격 조직에서 상술 한 단점 중요한 통계는 2,3- 관찰되었다 극복하기 위해 제안되었다. 그러나 이러한 분석은 여전히 존재 하나 심각한 제한 : 세포는 세포가 3 차원 구조를 제시 생체 내에서 관찰되는 것과 대조적으로 매우 확산 표현형을 나타낸다. 배양 방법과 관련이 유물은 강화 된 스트레스 섬유 1,4,5으로 비 생리적 기능을 실행할 수 있습니다. 2D는 6,7 환경에 비해 입체 세포 배양 분석은 여러 장점을 제공한다. 그들은 생리 학적 관련성, 그리고 결과에 따라서 의미가있다. 예를 들어, 히드로 겔에 포함 된 세포는 3 차원 형상의 구조를 표시하지만, 그 모폴로지는 또 다른 하나의 셀에 8,9 다르다. 그러나, 그들의 모폴로지는 심사 응용 프로그램을 복잡하게 다른 하나의 셀에서 차이가있다. 또 다른 전략은 단일 포함하는 것입니다미세 공동 10, 11 셀. 셀의 위치, 모양, 극성 및 내부 세포 조직은 정상화 될 수 있습니다. 세포에 3D와 같은 아키텍처를 제공하는 외에, 마이크로 공동 또한 높은 콘텐츠 스크리닝 연구 10,12-14 수 있습니다; 단셀은 마이크로 어레이 및 세포 내 소기관으로 주문할 수 및 진화가 동시에 관찰 할 수있다. 이 규칙은 세포의 낮은 수 더 나은 시간 / 공간 해상도가 좋은 통계를 제공합니다. 유용한 화합물은 안정적으로 쉽게 식별 할 수 있습니다. 본 연구에서는 높은 콘텐츠 스크리닝 응용 10,12,13 대한 제조 및 새로운 3 차원 형상의 단일 세포 배양 시스템의 애플리케이션을 보여준다. 상기 장치는 탄성 중합체 마이크로 공동 (105 공동 / cm 2), 신조어 &#39;eggcups&#39;(EC)의 배열로 구성된다. 이 작품의 크기 및 EC의 총 부피는 개별 NIH3T3과 헬라 세포의 전형적인 볼륨에 최적화되어 있습니다세포 분열시. 원통형 - - 공동의 형태는 활성 프로세스의 시각화를위한 제대로 동양 세포 형태에 선택됩니다. 15,16 커버 슬립 복제 성형 유리에 부착 된 얇은 폴리 디메틸 실록산 (PDMS) 층 상으로 패턴 EC의 배열을 사용한다. 세포를 원심 분리에 의해 EC에 도입된다. 우리는 2D에서 동일한 세포에 비해 여기 관찰 및 3D (EC)의 세포 소기관 (액틴 스트레스 섬유, 골지 장치 및 핵)의 정상화를보고 (평면)의 표면. 우리는 또한 세포의 유사 분열 17시 cytokinetic 액토 마이 오신 링의 폐쇄로 활동 역학 과정의 관찰을보고합니다. 마지막으로, 우리는 신진 효모, 분열 효모와 C.와 같은 단단한 벽, 다른 시스템에서이 방법의 결과를 보여 모델 시스템의 넓은 범위에가는 방법의 적용 가능성을 확인 간스 배아. 우리는 옆에 상세하고 철저한 페이지를 표시순서 rotocol는 제작 및 3D 미세에 대한 &#39;eggcups&#39;을 적용합니다. 우리의 접근 방식은 간단하고 클린 룸을 필요로하지 않습니다. 우리는이 새로운 방법론은 배양 접시의 교체에, 약물 스크리닝 분석 및 맞춤 의학에 특히 흥미로운 일이 될 것으로 예상된다. 마지막으로, 우리의 장치는 암 (18) 또는 기초 연구 (19), 예를 들어, 외부 자극에 세포 응답의 분포를 연구하는데 도움이 될 것입니다.

<table><tbody><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;0 (ultrapure)</td><td>Millipore</td><td>-</td><td>Use always fresh water.</td></tr><tr><td>Parafilm (plastic film)</td><td>Bemis</td><td>PM-999</td><td>Adhere Parafilm to the lab bench using some water droplets and ensure a perfect surface flatness.</td></tr><tr><td>Photo-mask</td><td>Selba</td><td>-</td><td>&lt;a href=&quot;http://www.selba.ch/&quot; name=&quot;RANGE!D4&quot;&gt;http://www.selba.ch&lt;/a&gt;</td></tr><tr><td>Silicon wafer</td><td>Siltronix</td><td>-</td><td>&lt;a href=&quot;http://www.siltronix.com/&quot;&gt;http://www.siltronix.com/&lt;/a&gt;</td></tr><tr><td>SU-8 photoresist</td><td>MicroChem</td><td>2000 series</td><td>&lt;a href=&quot;http://www.microchem.com/Prod-SU82000.htm&quot;&gt;http://www.microchem.com/Prod-SU82000.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>SU-8 developer</td><td>MicroChem</td><td>-</td><td>&lt;a href=&quot;http://microchem.com/Prod-Ancillaries.htm&quot;&gt;http://microchem.com/Prod-Ancillaries.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>19030</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Sigmacote (siliconizing reagent )</td><td>Sigma-Aldrich</td><td>SL2-25ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>harmful, working in a fumehood is required; check the data sheet from the manufacturer for more information.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Chlorotrimethylsilane (TMCS)</td><td>Sigma-Aldrich</td><td>386529-100ML</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/386529?lang=fr&amp;amp;region=FR&amp;nbsp;&amp;nbsp;</td></tr><tr><td colspan="4">TMCS produces acute inhalation and dermal toxicity, and is highly flammable (with ignition flashback able to occur across considerable distances), consequently it should be used in a fume cupboard away from sources of ignition</td></tr><tr><td>Nitrile gloves</td><td>Kleenguard</td><td>57372</td><td>&lt;a href=&quot;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&quot;&gt;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips #0</td><td>Knittel glass</td><td>KN00010022593</td><td>&lt;a href=&quot;http://www.knittelglass.com/index_e.htm&quot;&gt;http://www.knittelglass.com/index_e.htm&lt;/a&gt;</td></tr><tr><td>Very fragile. Manipulate gently.</td><td></td><td></td><td></td></tr><tr><td>Sharp straight tweezers</td><td>SPI</td><td>0WSSS-XD</td><td>http://www.2spi.com/catalog/tweezers/t/elec7</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>http://www.bdbiosciences.com/cellculture/tubes/features/index.jsp</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td>&lt;a href=&quot;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&quot;&gt;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>The package contains both PDMS base and curing agent. Similar elastomers are available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Microscope glass slides</td><td>Dutscher</td><td>100001</td><td>&lt;a href=&quot;http://www.dutscher.com/frontoffice/search&quot;&gt;http://www.dutscher.com/frontoffice/search&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>DMEM high-glucose medium</td><td>Fisher Scientific</td><td>41965-039</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Bovine calf serum</td><td>Sigma-Aldrich</td><td>C8056-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Fisher Scientific</td><td>25200-072</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS 1x</td><td>Fisher Scientific</td><td>14200-067</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS is at 10x and should be diluted to 1x using ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>L-15 medium</td><td>Fisher Scientific</td><td>21083-027</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Medium for atmospheres without CO&lt;sub&gt;2&lt;/sub&gt; control</td><td></td><td></td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F1141-5MG</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Penicillin &amp;amp; Streptomycin</td><td>Fisher Scientific</td><td>15140-122</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&lt;/a&gt;</td></tr><tr><td>Petri dish P35</td><td>Greiner</td><td>627102</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/en/row/articles/catalogue/article/144_11/12885/&lt;/a&gt;</td></tr><tr><td>Petri dish P60</td><td>Greiner</td><td>628163</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/145_8_bl/24872/&lt;/a&gt;</td></tr><tr><td>Petri dish P94</td><td>Greiner</td><td>633179</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/146_8_bl/24882/&lt;/a&gt;</td></tr><tr><td>Paraformaldehyde 3 %</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>Harmful in-particular for the eyes, working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>Triton 0.5 %</td><td>Sigma-Aldrich</td><td>93443-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Phallodin-Green Fluorescent Alexa Fluor 488</td><td>InVitrogen</td><td>A12379</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>dissolve powder in 1.5 ml methanol</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647</td><td>InVitrogen</td><td>A21245</td><td>1:200 dilution in PBS 1x</td></tr><tr><td>rabbit polyclonal anti-Giantin</td><td>Abcam</td><td>ab24586</td><td>1:500 dilution in PBS 1x</td></tr><tr><td>&lt;a href=&quot;http://www.abcam.com/giantin-antibody-ab24586.html&quot;&gt;http://www.abcam.com/giantin-antibody-ab24586.html&amp;nbsp;&lt;/a&gt;</td><td></td><td></td><td></td></tr><tr><td>rabbit anti-anillin</td><td>Courtesy of M. Glotzer, Published in Piekny, A. J. &amp;amp; Glotzer, M. Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Current biology 18, 30&amp;ndash;6 (2008).</td><td></td><td>1:500 dilution in PBS 1x</td></tr><tr><td>Anti-phosphotyrosine</td><td>Transduction Lab</td><td>610000</td><td>http://www.bdbiosciences.com/ptProduct.jsp?ccn=610000</td></tr><tr><td>Cy3 goat anti-rabbit&amp;nbsp;</td><td>Jackson Immunoresearch</td><td>111-166-047</td><td>&lt;a href=&quot;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&quot;&gt;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1:1,000 dilution in PBS 1x</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417&amp;nbsp;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1 mg/ml for 1 min</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G2025</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>HeLa cells</td><td>-</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>NIH3T3 cells</td><td>ATCC</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>Fission yeast&amp;nbsp;</td><td>-</td><td>-</td><td>For details on strains, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; worms</td><td>-</td><td>-</td><td>For details, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>YES (Agar) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-732</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>YES (Media) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-522</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow the instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>EMM (Media)</td><td>MP Biomedicals</td><td>4110-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>Filter sterilized EMM (Media) - Only for imaging</td><td>MP Biomedicals</td><td>4110-012</td><td>For preparation: follow instructions as given on the box. Filter sterilize the media using a 0.22 &amp;micro;m filter instead of autoclaving. This gives transparency to the media and reduces the autofluorescence.</td></tr><tr><td>Supplements (for EMM)</td><td>MP Biomedicals</td><td>4104-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&lt;/a&gt;</td></tr><tr><td>&amp;nbsp;(Add 225 mg/L into the EMM media before autoclaving or filtering)</td><td></td><td></td><td></td></tr><tr><td>Stericup and Steritop Vaccum driven sterile filters</td><td>Millipore</td><td>-</td><td>&lt;a href=&quot;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&quot;&gt;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&lt;/a&gt;</td></tr></tbody></table>

ordering single cells and single embryos in 3d confinement: a new device for high content screening

생물 세포는 일반적으로 평면 (2D) 표면에서 관찰된다. 이 조건은 생리적없고, 표현형 및 모양이 매우 다양하다. 이러한 환경에서 세포 기반 스크리닝 따라서 심각한 한계를 가지고 : 세포 소기관은 이종 및 / 또는 특정 세포 소기관이 제대로 시각화 할 수없는 극단적 인 표현형, 세포의 형태 학적 및 크기를 표시합니다. 또한, 생체 내의 세포는 3 차원 환경에서의 위치; 이 상황에서, 세포는 주로하기 때문에 조직의 주변 세포 외 기질과의 상호 작용의 서로 다른 표현형을 보여줍니다. 표준화 및 세포 기반 검정을위한 생리 학적으로 관련 차원 환경에서 하나의 셀의 순서를 생성하기 위해, 여기에서 3D 관내 세포 배양 장치의 미세 가공 및 애플리케이션을보고한다. 이 장치는 마이크로 공동의 2 차원 배열 (일반적으로 10 5 공동 / cm 2), 단일 세포 또는 배아로 가득 각각 구성되어있다. 셀 포지기, 모양, 극성 및 내부 세포 조직은 3 차원 구조를 보여주는 다음 정규화된다. 우리는 커버 슬립에 부착 된 얇은 폴리 디메틸 실록산 상 &#39;eggcups&#39;(PDMS) 층 패턴 마이크로 공동의 배열을 복제 성형을 사용했다. 캐비티가 접착을 촉진하기 피브로넥틴으로 피복 하였다. 균체를 원심 분리에 의해 삽입 하였다. 충전 비율을 80 %까지 허용하는 각 시스템에 대해 최적화되었다. 배아 세포 생존이 확인되었다. 우리는 핵 및 골지 장치와 같은 세포 소기관의 시각화를 위해이 방법을 적용, 이러한 세포 유사 분열 동안 cytokinetic 반지의 폐쇄 활성 프로세스를 연구. 이 장치는 골지체와 핵 정렬에 대한 cytokinetic 링 폐쇄 및 압축 된 표현형 중에 이러한주기적인 축적과 미오신과 액틴의 불균질성과 같은 새로운 기능의 식별을 허용했다. 우리는 포유류 세포, 분열 효모, 출아 효모, C.하는 방법을 특징으로하는 엘레 w각각의 경우에 i 번째 특정 적응. 마지막으로,이 장치의 특성은 약물 스크리닝 분석 및 맞춤 의학에 특히 흥미합니다.

&#39;eggcups&#39;(EC)는 3 차원 환경에서 배향 배아 세포의 가시화를 허용하는 신규 한 고 함량의 스크리닝 방법이다. 또한, 표준 2 차원 (평면) 배양 물에서 관찰하기 어려운 몇몇 세포 과정은이 새로운 방법에 의해 관찰 할 수있다.도 1a (제 1 전술 한 프로토콜도 참조 EC 미세 가공하기위한 절차의 개요를 나타낸다 ). 상기 방법은도 1B. 간단한 빠르고 효율적인 특수 장비의 필요?...

Watch this Scientific Journal Video about Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening at JoVE.com

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

우리는 세포와 배아를 연구하는 장치와 새로운 방법을보고합니다. 단일 세포는 정확하게 마이크로 공동 어레이에 정렬됩니다. 그들의 3D 감금은 생리 학적 조건에서 발생하는 3D 환경을 향한 단계이며 소기관 방향을 수 있습니다. 세포의 모양을 제어함으로써,이 설정은 표준 분석에보고 된 변동성을 최소화 할 수 있습니다.

단일 세포 및 3D 감금 단일 배아 주문 : 하이 콘텐츠 스크리닝을위한 새로운 장치

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

ordering-single-cells-single-embryos-3d-confinement-new-device-for

Research

JoVE Journal

Bioengineering

8.5K 조회수.  Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg.  우리는 세포와 배아를 연구하는 장치와 새로운 방법을보고합니다. 단일 세포는 정확하게 마이크로 공동 어레이에 정렬됩니다. 그들의 3D 감금은 생리 학적 조건에서 발생하는 3D 환경을 향한 단계이며 소기관 방향을 수 있습니다. 세포의 모양을 제어함으로써,이 설정은 표준 분석에보고 된 변동성을 최소화 할 수 있습니다. 

비디오: Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

분리 및 Zebrafish의 배아에서 단일 세포의 특성

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

연구

발생학

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

최적화 된 Microinjection 및 Zebrafish 애벌레의 이미징 microstructured 장치

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

생체공학

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans 배아 개발을 분석하는 세미 고처리량 이미징 방법 및 데이터 시각화 툴킷

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

자동 고함량 분석기를 사용하여 수행된 살아있는 세포 현미경 검사법을 위한 Drosophila 배아 준비 및 미세 주입

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

반복 패턴 형성을 위한 하이브리드 젤 큐브 장치에 초기 3D 셀 클러스터 제어

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

화학 유인 그라데이션에 대 한 다중 세포 반응의 3D 분석

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

정확한 세포 표현과 정확한 단백질 국소화를 위한 세균 세포의 3차원 이미징

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

면역학 및 감염병학

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

정량적 이미징 기법을 이용한 이종 세포 집단의 차별화 및 특성 규명

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

생물학

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

단세포 분해능 3차원 알래기 알코노이드

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...