Damljanović Vesna, Lagerholm B Christoffer, Jacobson Ken
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Biotechniques. 2005 Dec;39(6):847-51. doi: 10.2144/000112026.
Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.
目前，越来越多的细胞机械转导研究采用由聚丙烯酰胺制成的薄凝胶形式的弹性基质。它们的多功能性取决于确保适当的凝胶硬度以及控制凝胶细胞外基质蛋白涂层的均匀性和几何形状的能力。从简要定量强调聚丙烯酰胺凝胶的弹性特性开始，我们提出了一种廉价且高度可重复的方法，用于用多种细胞外基质蛋白进行均匀涂层。我们使用一种还原剂水合肼，将聚丙烯酰胺中无反应性的酰胺基团修饰为具有高反应性的酰肼基团，该基团可与氧化蛋白中的醛基或酮基形成共价键。这种简单的共轭方法克服了先前使用的光可激活交联剂的局限性：由于照射不均匀导致涂层不均匀，以及微图案化的技术挑战性程序。正如我们在受限迁移过程中对细胞极性的研究中所展示的那样，这种共轭方法在通过手动微接触印刷小至5微米的蛋白质图案进行凝胶微图案化方面特别有效，并且能够进行许多以前由于成本高或难以控制蛋白质涂层而无法实现的受限细胞附着和迁移研究。
