Zepto plasma system

Micropatterning was performed using a deep-UV light technique to normalize cell shape and adhesion area, as previously described (Azioune et al, 2009 (link)). Glass coverslips (square 22 × 22 mm, 1.5, VWR; or round 25 mm, 1.5; Thermo Fisher Scientific) were activated with plasma (Zepto Plasma System, Diener Electronic) for 2 min. After plasma treatment, coverslips were incubated with 0.2 mg/ml PLL(20)-g [3,5]-PEG(2) (SuSoS) in 10 mM Hepes at pH 7.4, for 1 h, at RT. Coverslips were washed three times with water, and left to dry before being placed on a synthetic quartz photomask (Delta Mask), previously activated with deep-UV light (PSD-UV; Novascan Technologies) for 5 min, using 3 μl of Milli-Q water to seal it to the mask. The coverslips were then irradiated through the photomask with the UV lamp for 5 min and left to dry before being incubated with FBN (25 μg/ml; F1141; Sigma-Aldrich), in 100 mM NaHCO3 at pH 8.6, for 30 min, at RT. Whenever possible, 5 μg/ml Alexa Fluor 647–conjugated fibrinogen (Thermo Fisher Scientific) was added to the FBN mix in order to visualize the pattern surfaces. Cells were added to the freshly incubated coverslips and allowed to spread for 15 min, before removing excess cells and new culture medium was added, and cells were left to fully adhere for another 12–16 h.

Initially, surfaces were cut into 1.0 × 1.0 cm² substrates and cleaned with ethanol (analytical grade, POCH S.A., Gliwice, Poland) in an ultrasonic bath to remove organic contaminents. Afterwards, the investigated surfaces were exposed to oxygen radio frequency (RF) plasma (Zepto plasma system, 40 Hz, 100 W, Diener Electronic, Ebhausen, Germany, 15 min at 50 W). The high hydrophilic character of the substrates after the treatment using plasma indicates the formation of a significant amount of polar surface silanols important to manufacture the covalent attachment of modifiers to the investigated surfaces [36 ].

10-µm-width line micropatterns to control individual cell shape and adhesion pattern were produced as follows: glass coverslips (22 × 22 mm, no. 1.5, Van Waters and Rogers) were activated with plasma (Zepto Plasma System, Diener Electronic) for 1 min and incubated with 0.1 mg/ml of PLL(20)-g[3,5]-PEG(2) (SuSoS) in 10 mM Hepes at pH 7.4 for 1 h at room temperature. After rinsing and air-drying, the coverslips were placed on a synthetic quartz photomask (Delta Mask), previously activated with deep-UV light (PSD-UV, Novascan Technologies) for 5 min. 3 µl of MiliQ water was used to seal each coverslip to the mask. The coverslips were then irradiated through the photomask with the UV lamp for 5 min. Afterward, coverslips were incubated with 25 µg/ml of fibronectin (Sigma-Aldrich) and 5 µg/ml of Alexa 647–conjugated fibrinogen (Thermo Fisher Scientific) in 100 mM NaHCO₃ at pH 8.6 for 1 h at room temperature. Cells were seeded at a density of 50,000 cells/coverslip and allowed to spread for ~10–15 h before imaging. Nonattached cells were removed by changing the medium ~2–5 h after seeding.