Name: a quantitative evaluation of cell migration by the phagokinetic track motility assay
Uploaded: 2012-12-04T12:00:00
Description: Watch this Scientific Journal Video about A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay at JoVE.com

Эта работа была поддержана грантами от Национального института здоровья (AI050677, HD-051998, и GM103433), Малкольм Feist сердечно-сосудистых исследований в общении и Американской ассоциации сердца predoctoral стипендий (10PRE4200007).

1. Подготовка желатин покрытием Покровные <ol><li> Место кислотно-промытый покровные стекла (15 мм в диаметре) в стерильный пластиковый 100 мм блюдо (а). Поместите 8-9 покровных на блюдо и убедиться, что они не соприкасаются друг с другом или сторон блюдо .. </li></ol> Примечание:</stron...

<ol>
	<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+cell+motility+during+embryogenesis.">The control of cell motility during embryogenesis.</a> Cancer Metastasis Rev. 4, 59-79 (1985).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+and+go+traffic+to+tune+T+cell+responses.">Stop and go traffic to tune T cell responses.</a> Immunity. 21, 305-314 (2004).</li><li>Mutsaers, S. E., Bishop, J. E., McGrouther, G., Laurent, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tissue+repair:+from+wound+healing+to+fibrosis.">Mechanisms of tissue repair: from wound healing to fibrosis.</a> Int. J. Biochem. 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Biol. Chem. 279, 54862-54871 (2004).</li><li>Ando, Y., Jensen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+insulin-like+growth+factor+I+enhance+keratinocyte+migration.">Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration.</a> J. Invest. Dermatol. 100, 633-639 (1993).</li><li>Todt, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tumor+necrosis+factor-alpha+on+human+trophoblast+cell+adhesion+and+motility.">Effects of tumor necrosis factor-alpha on human trophoblast cell adhesion and motility.</a> Am. J. Reprod. Immunol. 36, 65-71 (1996).</li><li>McAuslan, B. 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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=Roles+of+phosphatidylinositol+3-kinase+and+NF-kappaB+in+human+cytomegalovirus-mediated+monocyte+diapedesis+and+adhesion:+strategy+for+viral+persistence.">Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence.</a> J. Virol. 81, 7683-7694 (2007).</li><li>Bentz, G. L., Yurochko, 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=Human+CMV+infection+of+endothelial+cells+induces+an+angiogenic+response+through+viral+binding+to+EGF+receptor+and+beta1+and+beta3+integrins.">Human CMV infection of endothelial cells induces an angiogenic response through viral binding to EGF receptor and beta1 and beta3 integrins.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 5531-5536 (2008).</li><li>Chan, G., Nogalski, M. T., Yurochko, 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=Activation+of+EGFR+on+monocytes+is+required+for+human+cytomegalovirus+entry+and+mediates+cellular+motility.">Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 22369-22374 (2009).</li><li>Nogalski, M. T., Chan, G., Stevenson, E. V., Gray, S., Yurochko, 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=HCMV-Regulated+Paxillin+in+Monocytes+Links+Cellular+Pathogenic+Motility+to+the+Process+of+Viral+Entry.">HCMV-Regulated Paxillin in Monocytes Links Cellular Pathogenic Motility to the Process of Viral Entry.</a> J. Virol. 85, 1360-1369 (2011).</li><li>Turkevich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+nucleation+and+growth+processes+in+the+synthesis+of+colloidal+gold.">A study of the nucleation and growth processes in the synthesis of colloidal gold.</a> Discuss. Faraday. Soc. 11, 55-75 (1951).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size+and+sol+stability+in+mental+colloids.">Particle size and sol stability in mental colloids.</a> Colloid &#38; Polymer Science. 250, 736-741 (1972).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+nucleation+for+the+regulation+of+the+particle+size+in+monodisperse+gold+suspensions.">Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions.</a> Phys. Sci. 241, 20-22 (1973).</li><li>Zetter, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+capillary+endothelial+cell+migration.">Assay of capillary endothelial cell migration.</a> Methods Enzymol. 147, 135-144 (1987).</li><li>Scott, W. N., McCool, K., Nelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+the+production+of+gold+colloid+monolayers+for+use+in+the+phagokinetic+track+assay+for+cell+motility.">Improved method for the production of gold colloid monolayers for use in the phagokinetic track assay for cell motility.</a> Anal. Biochem. 287, 343-344 (2000).</li><li>Wang, S. Y., Mak, K. L., Chen, L. Y., Chou, M. P., Ho, C. 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Y., Chenn, 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+cell+behavior+screen:+identification,+sorting,+and+enrichment+of+cells+based+on+motility.">A cell behavior screen: identification, sorting, and enrichment of cells based on motility.</a> BMC Cell Biol. 6, 14 (2005).</li><li>Pan, 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=Size-dependent+cytotoxicity+of+gold+nanoparticles.">Size-dependent cytotoxicity of gold nanoparticles.</a> Small. 3, 1941-1949 (2007).</li><li>Rodriguez, L. G., Wu, X., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+assay.">Wound-healing assay.</a> Methods Mol. 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Phagokinetic анализа подвижности дорожки представленные в этой статье является простой и очень эффективный метод для количественного анализа миграции клеток. Потому что несколько типов клеток могут быть проанализированы 9-17, этот метод имеет потенциал широкого использования несколь...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> Покровные стекла (15 мм) </td><td> Fisher Scientific </td><td> 12-545-83 </td><td></td></tr><tr><td> Желатин 300 Bloom </td><td> Sigma-Aldrich </td><td> G-1890 </td><td></td></tr><tr><td> Tetrachloroauric Trihydrate кислота </td><td> Фишер химической </td><td> G54-1 </td><td> 14,5 мм (окончательное решение рабочей) </td></tr><tr><td> Цитрат натрия </td><td> Fisher Scientific </td><td> BP327-500 </td><td> 0,5% (окончательное решение рабочей) </td></tr><tr><td> Параформальдегид </td><td> Fisher Scientific </td><td> O4042 </td><td> 3% (окончательное решение рабочей) </td></tr><tr><td> 100 мм блюдо культуры ткани </td><td> Sarstedt </td><td> 83,1802 </td><td></td></tr><tr><td> 12-луночные планшеты </td><td> Fisher Scientific </td><td> 08-772-29 </td><td></td></tr><tr><td> 24-луночные планшеты </td><td> Fisher Scientific </td><td> 07-200-84 </td><td></td></tr><tr><td> Techne печь Hybridiser HB-1D </td><td> LabPlanet </td><td> 2040500 </td><td> Стандартные печи лаборатория будет достаточно </td></tr><tr><td> 10 мл Пипетки серологические </td><td> Sarstedt </td><td> 86.1254.001 </td><td></td></tr><tr><td> Pipet-Aid Filler / диспенсер </td><td> Драммонд </td><td> 13-681-15 </td><td></td></tr><tr><td> P200 Single-Channel Руководство пипетки </td><td> Rainin </td><td> PR-200 </td><td></td></tr><tr><td> 200 советов мл Барьер </td><td> CLP </td><td> BT200 </td><td></td></tr><tr><td> ImageJ программного обеспечения </td><td> <a href="http://rsb.info.nih.gov/ij/" target="_blank">HTT</a>р :/ / rsb.info.nih.gov / ц / </td><td></td><td> Лицензия: Public Domain </td></tr><tr><td> Nikon Eclipse TE300 с Фотометрия CoolSNAPfx монохромный 12-битной CCD камеры </td><td> Nikon </td><td></td><td> Снято; наиболее сопоставимыми спецификации имеет Nikon Eclipse Ti, а нижний конец Nikon 80i будет подходящим, а также. Другие бренды также обеспечить сопоставимые микроскопов. </td></tr><tr><td></td><td></td><td></td><td> Примечание: реагенты и оборудование, перечисленные ниже, были использованы нами в различных исследованиях. Другие материалы, поставщики, реактивы и оборудование можно использовать, пока они имеют сходные характеристики. </td></tr></tbody></table>

a quantitative evaluation of cell migration by the phagokinetic track motility assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

Показанный пример фотографии, сделанные под световым микроскопом показывает трек область очищена одна ячейка (моноцитов из наших экспериментах показано на рисунке 2). Non-подвижных клеток создают характерный небольшие, овальной или круг формы участки вокруг себя указывает на ?...

Watch this Scientific Journal Video about A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay at JoVE.com

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Phagokinetic анализа трек подвижности является метод, используемый для оценки движения клеток. В частности, анализ мер хемокинез (случайный подвижность клеток) с течением времени в количественном выражении. Анализ использует способность клеток для создания измеримых отслеживать их движение на коллоидное золото покрытием покровные.

Количественные оценки миграции клеток в Phagokinetic анализа подвижности трек

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay для оценки патогена индуцированной нейтрофил трансэпителиальной миграции

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Количественная оценка миграции нейтрофилов человека во культурный мочевого эпителия

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Конформационный Оценка ВИЧ-1 тримерных гликопротеинов с помощью мобильного основе ELISA Пробирной

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Высокая пропускная способность Количественный реальном времени RT-PCR анализа для определения профилей экспрессии типов I и III интерферона подтипов

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Качественный и количественный анализы для обнаружения и определения характеристик белка Противомикробные

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...