N n n n tetramethylethylenediamine temed

Polyacrylamide (PA) gels were prepared as previously described [16 (link),17 (link)]. Briefly, 40% w/v acrylamide and 2% w/v bis-acrylamide solutions (Bio-Rad, Hercules, CA) were mixed with 10 mM HEPES buffer. PA gels with different stiffness were obtained by varying the final concentrations of acrylamide solution (3-7.5%) and bis-acrylamide cross-linker (0.03-0.6%). To polymerize the solution, 2.5 μL of 10% w/v ammonium persulfate (Bio-Rad) and 0.25 μl of N,N,N’,N’-Tetramethylethylenediamine (TEMED; Bio-Rad) were added with the solution to yield a final volume of 500 μl. After polymerization, sulfo-SANPAH (N-Sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate; Pierce Biotechnology, Rockford, IL), a photo-activating cross-linker, was used to crosslink the extracellular matrix molecules onto the gel surface. The powder of sulfo-SANPAH was dissolved in 10 mM HEPES buffer containing 0.5% DMSO and the sulfo-SANPAH solution (0.5 mg/ml) was added on top of the PA gel. Gel dishes were placed at a distance of ~15 cm from the UV light of the hood for 6 min and rinsed three times with 10 mM HEPES buffer for 10 min. A 200 μl of collagen type I solution (40 ug/ml) (BD Sciences, Bedford, MA) was incubated on top of the sulfo-SANPAH-coated PA gels at 37 °C overnight to deposit collagen for subsequent cell seeding.