使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响
Summary
可以仿照下层刚度对细胞功能的影响<em>在体外</em>使用不同的符合标准的聚丙烯酰胺凝胶。
Abstract
组织刚度，是一个重要的细胞功能因素，并组织刚度的变化通常与纤维化，癌症和心血管疾病的1-11的关联。传统的细胞研究细胞功能的生物方法涉及一个刚性基质上培养细胞（塑料餐具或盖玻片），它可以不考虑弹性ECM或流脑组织之间的刚度变化的影响。 在体内组织遵守的条件，在体外模型，我们和其他人使用ECM涂水凝胶。在我们的实验室，是基于水凝胶聚丙烯酰胺可以模仿生物12见过的组织符合。除了3 APTMS孵化用NaOH产生“反应”的盖玻片。戊二醛是用于交叉连接的3 APTMS和聚丙烯酰胺凝胶。用于水凝胶的聚合丙烯酰胺（AC），双丙烯酰胺（BIS – AC），过硫酸铵溶液。 N -羟基琥珀酰亚胺（NHS）是纳入交联的ECM蛋白凝胶交流解决方案。聚合凝胶，凝胶表面涂有流脑的首选，如纤维连接蛋白，vitronectin，胶原蛋白等蛋白质
水凝胶的硬度，可以由流变或原子力显微镜（AFM）和由不同的解决方案中的12的交流和/或双AC百分比调整。在这种方式下，下层刚度可以匹配生物组织的刚度，也可使用流变或AFM量化。细胞可以接种这些水凝胶和培养根据所需的实验条件。成像的细胞和分子生物学分析他们的恢复很简单。在这篇文章中，我们定义为那些有<3000帕斯卡与E和僵硬的组织下层/> 20,000帕斯卡的弹性模量（E）软下层。
Protocol
Discussion
凝胶聚合过程的一个关键因素是，以避免气泡的形成，这将使细胞绑定到ECM涂层凝胶本身，而不是玻璃盖玻片。这是可以预防的仔细吹打后震荡和可视化，确保无气泡已成为被困在凝胶的聚合解决方案。我们始终建议准备额外的“反应”盖玻片和凝胶，以确保有足够的实验。
凝胶用PBS冲洗时，应特别注意支付。吸痰过程中必须避免与水凝胶本身，因为它可能被抓住，撕裂的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
工作是我们的实验室是由美国国立卫生研究院的赞助支持。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Glutaraldehyde, 70% | Sigma-Aldrich | G7776 | Store at -20°C | |
| 3-APTMS (3-Aminopropyltrimethosysilane 97%) | Sigma-Aldrich | 281778 | Store at room temperature | |
| SurfaSil Siliconizing Fluid | Thermo Scientific | 42800 | Store at room temperature | |
| NHS (N-hydroxysucinimide Ester) | Sigma-Aldrich | A-8060 | Store at 4°C Replace monthly | |
| Albumin, bovine serum, essentially fatty acid free | Sigma-Aldrich | A6003-100G | Store at 4°C | |
| Coverslips (25mm) | Fisher Scientific | 12-545-86 25 Cir 1D | ||
| Coverslips (18mm) | Fisher Scientific | 12-545-84 18 Cir 1D |
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