Poly n isopropylacrylamide

Glass slides (2.2 × 2.2 cm) were washed with detergent and MilliQ (Millipore, US) water. After drying, the slides were covered with poly(N-isopropylacrylamide) (Sigma-Aldrich, St. Louis, MO) dissolved in ethanol (6% w/v). The slides were etched via a shadow mask by oxygen plasma for 5 min at 200 W of power. The reverse transfection protocol was previously described. In brief, 3 µL OptiMEM (Invitrogen) containing sucrose and 2.5 µL Lipofectamine 3000 (Invitrogen) were transferred to each tube and mixed thoroughly. Then, 2 µg siRNA/1 µg of plasmid/chemicals/proteins were added to each tube, and the mixture was incubated for 20 min at room temperature. Finally, 7.25 µL of a 0.2 % (w/v) gelatin (Type B; Sigma-Aldrich) solution was added to each tube and mixed thoroughly. After UV sterilization, the reverse transfection reagent was printed on the chip using a nanodispenser (Phoenix; Art Robbins Instruments, Sunnyvale, CA). Next, the slides were fixed in a 6-well plate with melted wax. Approximately 3 mL of 37 °C medium containing 5 × 10 5 cells was transferred to each well. Approximately 24 to 48 h later, the dishes were moved at room temperature for 5 min and were washed with phosphate-buffered saline (PBS) three times to ensure the total removal of the polymer.