प्रोटीन और सेल बन्दिश के लिए दो आयामी नमूनों Substrates बनाना
Summary
स्व इकट्ठे monolayers (SAMs) सोने पर लंबी श्रृंखला alkane thiols गठन से अच्छी तरह से परिभाषित प्रोटीन पैटर्न और सेल कारावास के गठन के लिए substrates प्रदान करते हैं. Hexadecanethiol के microcontact मुद्रण polydimethylsiloxane (PDMS) के साथ backfilling बाद स्टांप का उपयोग कर एक glycol समाप्त alkane thiol monomer के एक पैटर्न का उत्पादन जहां प्रोटीन और कोशिकाओं को केवल मुद्रांकित hexadecanethiol क्षेत्र सोखना.
Abstract
Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10
Protocol
Discussion
PDMS स्टांप निर्माण के लिए इस्तेमाल किया मास्टर के lithographic उत्पादन में मुद्दों का एक नंबर पैदा कर सकते हैं. अपूर्ण फोटो अस्पष्ट और अस्पष्ट पैटर्न और विरोध में लिपटे बढ़े या लापता सुविधाओं में वफ़र परिणामों …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम वाशिंगटन विश्वविद्यालय जिसका सामूहिक ज्ञान के इस प्रोटोकॉल संभव बनाया है में पूरे Maurer समूह स्वीकार करना चाहते हैं. मानसिक स्वास्थ्य के राष्ट्रीय संस्थान (1R01MH085495) द्वारा इस कार्य के लिए अनुदान उपलब्ध कराया जाता है.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Silicon wafer | Wafer Reclaim Services | 2 inch | |
| Spin coater/hot plate | Brewer Science | Cee 200CB Spin-Bake System | |
| AZ9245 Photoresist | Mays Chemical Company | 105880034-1160 | |
| Direct-write photolithography system | Microtech s.r.l. | LW325 LaserWriter System | |
| Mask Aligner | HTG | 3HR | |
| AZ 400K Developer | Mays Chemical Company | 105880018-1160 | |
| Sylgard 182 Silicone Elastomer Kit | Dow Corning | ||
| 25 mm no. 1 round glass coverslips | VWR | 16004-310 | |
| Plasma Oxidizer | Diener | Femto | |
| Titanium pieces Kamis | Incorporated | 99.95% pure | |
| Gold pellets | Kamis Incorporated | 99.999% pure | |
| Electron-beam evaporator | Kurt J. Lesker | PVD 75 Thin Film Deposition System | with electron-beam accessory |
| Hexadecanethiol | Alfa Aesar | A11362 | |
| 1-mercaptoundec-11-yl)tetra(ethyleneglycol) | Sigma Aldrich | 674508 | |
| Ethanol | Pharmco-aaper | 111000200 | 200 proof, absolute |
| Parafilm | VWR | 52858-000 | |
| DPBS | VWR | 4500-434 | Without calcium and magnesium |
| Mouse Laminin I | VWR | 95036-762 | |
| Human Plasma Fibronectin | Invitrogen | 33016-015 | |
| AlexaFluor® 647 carboxylic acid, succinimidyl ester | Invitrogen | A-20006 | |
| MitoTracker Red 580 | Invitrogen | M22425 | |
| AlexaFluor® 350 carboxylic acid, succinimidyl ester | Invitrogen | A-10168 | |
| Anti-laminin antibody | Fisher Scientific | AB2034MI |
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