Pll g peg

Patterned polyacrylamide hydrogels were prepared following the guidelines previously described in Vignaud et al. (2014) (link). A quartz photomask was first cleaned for 3.5 min with oxygen plasma (AST product, 300 W) at 200 W. Micropatterns were then incubated with 0.1 mg/ml PLL-g-PEG (JenKem Technology ZL187P072) in 10 mM HEPES, pH 7.4, for 30 min. After dewetting, the mask was exposed under deep UV for 5 min. Next, micropatterns on the mask were incubated with a mix of 10 µg/ml fibronectin (#F1141, Sigma) and 20 µg/ml fibrinogen-Alexa-Fluor-647 conjugate (#F35200, Invitrogen) in 100 mM sodium bicarbonate buffer, pH 8.4, for 30 min. A mix of acrylamide (8%) and bis-acrylamide solution (0.264%; Sigma) corresponding to a theoretical Young modulus of 19.66 kPa was degassed for approximately 30 min, mixed with 0.2 µm PLL-PEG covalently coated fluorescent beads (Fluorosphere #F8810, Life Technologies), and sonicated before addition of APS and TEMED. A quantity of 25 µl of that solution was added on the micropatterned photomask, covered with a silanized coverslip (Silane, #M6514, Sigma), and allowed to polymerize for 25 min before being gently detached in the presence of sodium bicarbonate buffer. Micropatterns were stored overnight in sodium bicarbonate buffer at 4°C before cells were plated.