جهاز لأداء الهجرة خلية / شفاء الجروح في لوحة 96-حسنا
Summary
Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.
Abstract
الهجرة خلية / اصابة الفحص هو الطريقة المستخدمة عادة لدراسة الهجرة الخلية والعمليات الحيوية الأخرى، مثل الأوعية الدموية والانبثاث ورم. في هذا الاختبار، تزرع الخلايا لتشكيل أحادي الطبقة متموجة ويتم إنشاء الجرح الميكانيكية بواسطة خدش مع الجهاز. ثم يمكن رصدها معدل الهجرة من الخلايا باتجاه منطقة الجرداء بواسطة التصوير. تم تصميم wounder الميكانيكية 8 قنوات لمعالجة معظم المشاكل المرتبطة فحص الهجرة الخلية. أولا، wounder لدينا يمكن تعقيمها بسهولة عن طريق التعقيم أو مع المطهرات الشائعة. ثانيا، دبابيس قابل للتعديل الفردية تسمح الاتصال حتى مع لوحة الثقافة الخلية بحيث يمكن إنشاء الجروح الحادة وقابلة للتكرار. ثالثا، قضبان توجيه على كلا الجانبين من wounder ضمان موقف جرح ثابت في كل بئر. استخدام المتاح نصائح ماصة بلاستيكية للإصابة يمكن أن يزيد من توفير معالجة أفضل للwounder وكذلك لتقليل عبر contaminatأيون. في الختام، يمكن لنا wounder خلية توفر الباحثين مع جهاز سهل الاستخدام وقابلة للتكرار لأداء الفحص الهجرة الخلية باستخدام معيار لوحة ثقافة 96-جيدا.
Introduction
Cell migration plays an important role in cellular processes, such as embryogenesis, neurogenesis, angiogenesis, wound healing, mucosal repairing, epithelial-mesenchymal-transitions under normal development and disease situation1. It is a complicated process which involves the coordination of numerous inter- and intra-cellular events including signaling molecule interactions, cell polarization, cytoskeletal reorganization, matrix remodeling, membrane protrusion and dynamic cell-cell adhesion modulation2. By studying cell migration, the discovery and validation of chemicals or biomolecules leading to cell movement and its related biochemical pathways can be determined. This fundamental process can be used beneficially as a proxy for various applications, such as disease treatment and drug targeting.
The wounding assay is one of the cell migration assays3. Here, an individual sample of cells will be partially removed by mechanical means to produce a denuded area where cells will migrate to cover the area. The percentage of recovery at a particular time point will be monitored. When handling large number of samples, issues such as the experimental cost and cross-contamination within samples can be problematic. Although many commercial tools and assays are available for studying cell migration4,5,6, many of them required expensive and sophisticated equipment and improvements are still needed. For these reasons, an 8-channel mechanical cell wounder (Figure 1) is developed.
Our 8-channel mechanical cell wounder has incorporated several unique features in solving the above-mentioned problems. It provides the flexibility to perform cell migration/wounding assays in the 96-well culture plate format. The adjustable guiding bar design ensures that the scratch area is in the central position of each well. Also, the height of the guiding bars can be adjusted so that the wounder is applicable for various brands of culture plates. Finally, the adjustable wounding pin design enables an even contact of the pipette tips with the culture plate surface to achieve simultaneous and reproducible wounding7,8.
Protocol
Representative Results
Discussion
Our cell wounder has several unique features in solving the problems of traditional cell migration assays. The 8-channel mechanical cell wounder is made of high grade stainless steel (Steel 304) with a long lifespan that can be sterilized by autoclaving. Almost all commercial brands of 96-well culture plates available on the market can be used with this mechanical cell wounder because of the adjustable guiding bar. The design of the adjustable guiding bar also ensures that the scratching area is at the center of each wel…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Mr. Tam Po Leung, Mr. Wong Chi Kin and the technical staff of the Science Faculty Workshop, Hong Kong Baptist University, for their technical skills and advice in making the prototype of this wounder.
Materials
| 96-well cell culture plate | Nunc | 167008 | Other brands of 96-well cell culture plate can also be used |
| P10 pipette tips | Axygen | 301-03-051 | Short P10 pipette tip is more easy to create a clear wound |
| Wounder | R&P Technology Limited | ||
| Medium 199 | Sigma | M2520-1L | For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells. |
| Fetal bovine serum | Gibco | 26140079 | For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells. |
| Penicillin/Streptomycin | Gibco | 15140122 | For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells. |
| Heparin sodium salt from porcine intestinal mucosa | Sigma | H3393 | For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells. |
| Gelatin from bovine skin | Sigma | G9391 | For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells. |
Riferimenti
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