Name: a customizable chamber for measuring cell migration
Uploaded: 2017-03-12T14:00:00
Description: Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration Video at JoVE.com

الكتاب يقر البرنامج الإبداعي رسالتك جامعة كليمسون وجبهة الخلاص الوطني CBET1254609 لتوفير التمويل اللازم لهذا المشروع.

1. إعداد وثيقة غرفة متناهية لإنشاء التدرج تركيز <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع العفن قطعة الاكريليك <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصول على قطعة من الاكريليك من العرض المطلوب. عادة ما تستخدم ورقة 1 الاكريليك / 16 بوصة ...

<ol>
	<li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly+(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly (dimethylsiloxane) for biological studies.</a> Electrophoresis. 24 (21), 3563-3576 (2003).</li><li>Lin, 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=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4 (3), 164-167 (2004).</li><li>Darby, I., Hewitson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Differentiation+in+Wound+Healing+and+Fibrosis.">Fibroblast Differentiation in Wound Healing and Fibrosis.</a> Int Rev Cytol. 257, 143-179 (2007).</li><li>Thampatty, B. P., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+study+fibroblast+migration.">A new approach to study fibroblast migration.</a> Cell Motil Cytoskel. 64, 1-5 (2007).</li><li>Discher, D., Mooney, D., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324, 1673-1677 (2009).</li><li>Zengel, P., 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=&#181;-Slide+Chemotaxis:+A+new+chamber+for+long-term+chemotaxis+studies.">&#181;-Slide Chemotaxis: A new chamber for long-term chemotaxis studies.</a> BMC Cell Biol. , (2011).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+Chamber+Assay.">Boyden Chamber Assay.</a> Methods Molec Biol. 2, 15-22 (2005).</li><li>Safferling, K., 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=Wound+healing+revised:+A+novel+reepithelialization+mechanism+revealed+by+in+vitro+and+in+silico+models.">Wound healing revised: A novel reepithelialization mechanism revealed by in vitro and in silico models.</a> JCB. 203 (4), 691-709 (2013).</li><li>Saadi, W., 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+stable+concentration+gradients+in+2D+and+3D+environments+using+a+microfluidic+ladder+chamber.">Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.</a> Biomed Microdevices. 9, 627-635 (2007).</li><li>Cammer, M., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+Responses+by+Macrophages+to+a+Directional+Source+of+a+Cytokine+Delivered+by+a+Micropipette.">Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette.</a> Methods Mol Biol. , 125-135 (2014).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J Cell Sci. 99 (4), 769-775 (1991).</li><li>Wells, C. M., 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=Analysis+of+cell+migration+using+the+Dunn+chemotaxis+chamber+and+time-lapse+microscopy.">Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.</a> Methods Mol Biol. , 31-41 (2005).</li><li>Liang, C. C., Park, A. Y., 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=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat. Protoc. 2, 329-333 (2007).</li><li>Chen, Y. 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=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci. Rep. 5, (2015).</li><li>Wright, G. A., 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=On-chip+open+microfluidic+devices+for+chemotaxis+studies.">On-chip open microfluidic devices for chemotaxis studies.</a> Microsc. Microanal. 18 (04), 816-828 (2012).</li><li>Adler, M., Polinkovsky, M., Gutierrez, E., Groisman, A. <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+oxygen+gradients+with+arbitrary+shapes+in+a+microfluidic+device.">Generation of oxygen gradients with arbitrary shapes in a microfluidic device.</a> Lab Chip. 10 (3), 388-391 (2010).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+devices:+New+tools+for+studying+cancer+and+cancer+stem+cell+migration.">Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration.</a> Biomicrofluidics. 5 (1), 013412 (2011).</li><li>Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chambers+for+cell+migration+and+neuroscience+research.">Microfluidic chambers for cell migration and neuroscience research.</a> Microfluid Tech: Rev Protoc. , 167-177 (2006).</li><li>Tang, S. K., Whitesides, G. M., Fainman, Y., Lee, L., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic microfluidic and soft lithographic techniques. Optofluidics: Fundamentals, Devices and Applications. , (2010).</li><li>Taylor, A. 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=Microfluidic+multicompartment+device+for+neuroscience+research).">Microfluidic multicompartment device for neuroscience research).</a> Langmuir. 19 (5), 1551-1556 (2003).</li><li>Aidelberg, G., Goldshmidt, Y., Nachman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Device+for+Studying+Multiple+Distinct+Strains.">A Microfluidic Device for Studying Multiple Distinct Strains.</a> JoVE. (69), (2012).</li></ol>

ويمكن استخدام لدينا غرفة متناهية للعديد من الأغراض، بما في ذلك تحديد معدل هجرة الخلايا استجابة لعوامل النمو وchemoattractants وقياس معدل انتشار المخدرات من البوليمر. ومن الممكن الاستفادة من غرفة متناهية لدينا لزراعة الخلايا ووضع جاذب كيميائي في واحدة من نهاية الغرفة. تنمو ...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
		</tr>
		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

هجرة الخلايا هي جزء حيوي من الاستجابات المناعية، والنمو، والتئام الجروح. هجرة الخلية هي عملية معقدة تنطوي على تفاعلات بين الخلايا، المصفوفة خارج الخلية، والعوامل الكيميائية القابلة للذوبان وغير القابلة للذوبان (على سبيل المثال، chemoattractants). طرق معيارية لقياس الهجرة من الخلايا، مثل فحص غرفة بويدن، والعمل عن طريق عد الخلايا على جانبي الفاصل. هذه التقنيات هي سهلة الاستخدام. ومع ذلك، وأنها توفر القليل من التعديل الهندسي لمختلف التطبيقات. في المقابل، وأجهزة ميكروفلويديك يمكن استخدامها لمراقبة الهجرة الخلية مع التدرجات تركيز قابلة للتخصيص من العوامل القابلة للذوبان 1 و 2. ومع ذلك، يمكن أساليب لجعل فحوصات على microfluidics أساس أن يكون من الصعب معرفة. هنا، نحن تصف طريقة سهلة لإنشاء غرف زراعة الخلايا لقياس الهجرة الخلية ردا على التدرجات تركيز الكيميائية. لدينا ميل خليةغرايشن طريقة الغرفة يمكن أن تخلق مختلفة التدرجات تركيز الخطية لدراسة الهجرة خلية لمجموعة متنوعة من التطبيقات. هذه الطريقة سهلة نسبيا للاستخدام، وعادة ما يقوم بها طلاب المرحلة الجامعية. تم إنشاء غرفة متناهية عن طريق وضع إدراج الاكريليك على شكل غرفة متناهية النهائية جيدا في طبق بيتري. بعد ذلك، تم صب بولي (dimethylsiloxane) (PDMS) على رأس إدراج. سمح للPDMS لتتصلب ومن ثم تم إزالة إدراج. وهذا ما سمح لإنشاء الآبار في أي الشكل المطلوب أو الحجم. ويمكن إضافة الخلايا بعد ذلك إلى غرفة متناهية، ويمكن إضافة عوامل قابلة للذوبان في واحدة من الآبار عن طريق نقع كتلة الاغاروز في عامل المرجوة. يضاف كتلة الاغاروز إلى إحدى الآبار، والصور الوقت الفاصل يمكن اتخاذها للغرفة متناهية من أجل تحديد الهجرة الخلية. الاختلافات إلى هذا الأسلوب يمكن أن يتم لتطبيق معين، مما يجعل هذه الطريقة المهزومةHLY للتخصيص.

ويبين الشكل 2 حركة الجبهة الخلية عبر القناة ردا على التدرج من المصل البقري الجنين وضعت في الطرف المقابل من القناة من حيث مطلي الخلايا. يظهر أمام الخلية في 48 ساعة (الشكل 2A)، 72 ساعة (الشكل 2B)، 96 ساعة (الشكل 2C)، و 120 ساعة...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration Video at JoVE.com

A Customizable Chamber for Measuring Cell Migration Video (Video) | JoVE

تفاصيل هذا البروتوكول طريقة تخصيص لقياس الهجرة الخلية ردا على chemoattractants التي يمكن أن تستخدم أيضا لتحديد معدل انتشار المخدرات من البوليمر.

للدائرة للتخصيص لقياس الهجرة الخلية

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Research

JoVE Journal

Biology

A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

Measuring Exocytosis in Neurons Using FM Labeling Video (Video) | JoVE

قياس إيماس في الخلايا العصبية عن طريق وصفها وزير الخارجية

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber Video (Video) | JoVE

تحفيز الدماغ شريحة باستخدام شبكة ميكروفلويديك وستاندرد غرفة الإرواء

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc Video (Video) | JoVE

يعيش التصوير للهجرة الخلايا الدبقية في القرص العين تخيلي ذبابة الفاكهة

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap Video (Video) | JoVE

قياس صلابة انحناء الخلايا البكتيرية باستخدام فخ البصرية

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

Measuring Peptide Translocation into Large Unilamellar Vesicles Video (Video) | JoVE

قياس إزفاء الببتيد في الحويصلات الكبيرة Unilamellar

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration Video (Video) | JoVE

حيوي داخلي المجهري للدوران الأوعية الدقيقة في العضلات المشمرة ماوس لتحليل الهجرة الجذعية الطرفية المحمولة

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Single-cell Microinjection for Cell Communication Analysis Video (Video) | JoVE

حقن مكروي وحيدة الخلية لتحليل الاتصالات خلية

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

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Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

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The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

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محسن المقايسة الصفر للاختبار في المختبر للهجرة الخلية مع كاميرا بصرية الآلي

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...