Name: fabrication and use of microenvironment microarrays (mearrays)
Uploaded: 2012-10-11T12:00:00
Description: Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

ML поддерживается NIA (R00AG033176 и R01AG040081) и лаборатории, руководимой исследований и развития, Министерство энергетики США контракту № DE-AC02-05CH11231.

1. Подготовка Печать Субстраты Решение об использовании полидиметилсилоксана (PDMS)-покрытием или полиакриламида (ПА)-покрытием слайды зависит от важных параметров эксперимента. Модуль упругости обоих полимеров может быть настроен для имитации жесткости различных тканей...

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	<li>Bissell, M. J., Labarge, 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=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., 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=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Dev. Cell. 6, 483-495 (2004).</li><li>LaBarge, 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=Human+mammary+progenitor+cell+fate+decsions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decsions are products of interactions with combinatorial microenvironments.</a> Integrative Biology. 1, 70-79 (2009).</li><li>Kim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Annals of biomedical engineering. , (2012).</li><li>Boudou, T., Ohayon, J., Picart, C., Pettigrew, R. I., Tracqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+properties+of+polyacrylamide+gels:+implications+for+quantification+of+cellular+forces.">Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces.</a> Biorheology. 46, 191-205 (2009).</li><li>Tse, J. R., Engler, 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=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current protocols in cell biology. Chapter 10, Unit 10 (2010).</li><li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+matrix+microarray+for+probing+cellular+differentiation.">An extracellular matrix microarray for probing cellular differentiation.</a> Nat. Methods. 2, 119-125 (2005).</li><li>Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+regulation+of+human+neural+precursor+cell+differentiation+using+arrays+of+signaling+microenvironments.">Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.</a> Mol. Syst. Biol. 2, 37 (2006).</li></ol>

MEArray метод, представленный здесь дает функциональный анализ клеточных взаимодействиях и комбинаторной микроокружения 4. MEArray анализ сочетает в себе использование базовых технологий micropatterning, клеточной биологии, и микрочипов печати роботов и анализ устройств, которые доступны на...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> Предметные стекла 25 мм х 75 мм </td><td> VWR </td><td> 48311-600 </td><td></td></tr><tr><td> Стекло покровные (№ 1) 24 мм х 50 мм </td><td> VWR </td><td> 48393-241 </td><td></td></tr><tr><td> Окрашивание блюдо (или Coplan банку) </td><td> VWR </td><td> 25461-003 </td><td></td></tr><tr><td> Чашки Петри (15 см) </td><td> BD Falcon </td><td> 351058 </td><td></td></tr><tr><td> NaOH (1,0 н) </td><td> Sigma-Aldrich </td><td> S2567 </td><td></td></tr><tr><td> APES (&gt; 98% (3-аминопропил) триэтоксисилана) </td><td> Sigma-Aldrich </td><td> A3648 </td><td></td></tr><tr><td> Глутаральдегид </td><td> Sigma-Aldrich </td><td> G7651 </td><td> 50% в воде </td></tr><tr><td> APS (&gt; 98% персульфата аммония) </td><td> SIGMA-Aldrich </td><td> A3678 </td><td> Подготовить 10% рабочего раствора с DDH 2 O </td></tr><tr><td> TEMED (N, N, N &#39;, N&#39;-тетраметилэтилендиамин) </td><td> Sigma-Aldrich </td><td> T9281 </td><td></td></tr><tr><td> Акриламида (40%) </td><td> Sigma-Aldrich </td><td> A4058 </td><td></td></tr><tr><td> Бис-акриламид (2% вес / объем) </td><td> Фишер BioReagents </td><td> BP1404-250 </td><td></td></tr><tr><td> 0,45 мкм шприц фильтр 4-мм нейлон </td><td> Nalgene </td><td> 176-0045 </td><td></td></tr><tr><td> FITC </td><td> Sigma-Aldrich </td><td> F4274 </td><td></td></tr><tr><td> PDMS (полидиметилсилоксан) </td><td> Dow Corning </td><td> 3097358-1004 </td><td> Sylgard 184 Эластомер комплекта через Клеи Ellsworth </td></tr><tr><td> 2-х камерный слайды </td><td> NUNC </td><td> 177380 </td><td></td></tr><tr><td> Pluronic F108 </td><td> BASF </td><td> 30089186 </td><td></td></tr><tr><td> AquArium герметика </td><td> Dow Corning </td><td> DAP 00688 </td><td></td></tr><tr><td> Fluormount-G </td><td> Southern Biotech </td><td> 0100-01 </td><td></td></tr><tr><td> Одноразовые пластиковые стаканчики </td><td></td><td></td><td></td></tr><tr><td> Язык депрессоров </td><td></td><td></td><td></td></tr><tr><td> Нитриловые перчатки </td><td></td><td></td><td></td></tr><tr><td> Пластиковые боксы стекло микроскопа </td><td></td><td></td><td></td></tr><tr><td> Спин для нанесения покрытий </td><td> WS-400B-6NPP/LITE </td><td> Laurell Technologies Corporation </td><td></td></tr><tr><td> Печь </td><td></td><td></td><td></td></tr><tr><td> Цифровой плиты </td><td></td><td></td><td></td></tr><tr><td> 384-луночных планшетов </td><td></td><td></td><td> Совершенно подходит для робота микрочипов </td></tr><tr><td> Microarray печати робот </td><td></td><td></td><td></td></tr><tr><td> Перевернутый фазы и флуоресцентного микроскопа </td><td></td><td></td><td></td></tr><tr><td> Axon микрочипов сканеры </td><td> Molecular Devices </td><td></td><td> Несколько конфигураций существуют </td></tr></tbody></table>

fabrication and use of microenvironment microarrays (mearrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

Fabrication and Use of MicroEnvironment microArrays MEArrays

Комбинаторной функциональный метод скрининга для получения понимание воздействия молекулярного состава микросреды на клеточные функции описаны. Метод использует существующие микрочипов на основе технологии для создания массивов определены комбинаторной микросреды, которые поддерживают клеточной адгезии и функционального анализа.

Изготовление и использование микроокружения микрочипов (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

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Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

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