Single-Cell Transfection Analysis via LOP

To analyze the behaviour of a single cells transfected with full length WT and P229S and to see the effect of the mutation on a single cell in detail, we used the method of photoresist lift-off assisted patterning of ECM proteins (LOP). Previously, Moeller et al. described the detailed protocol for this method (67 (link)). Shortly, the coverslips (18 × 18 mm Paul Marienfeld GmbH & Co. KG Lauda-Königshofen Germany) were cleaned using acetone, isopropanol and MQH₂O, dried and treated with oxygen plasma for 2 min (Vision 320 RIE, Advanced Vacuum). The photoresist S1818 (Microchem, Westborough, Massachusetts, USA) was spin-coated on the coverslips using the standard contact photolithography and 2 μm thick resist layer was photopatterned (5.6–7.5 mW/cm² at 365 nm, OAI instruments). The ice-cream patterns were designed using a chrome mask. The S1818 structure on the coverslip was treated with oxygen plasma for 15 s at 80 W (PICO plasma cleaner; diener) followed by incubating the S1818 structure with 0.1 mg/ml PLL-g-PEG in PBS solution for 30 min at room temperature. The coverslip was then rinsed with MQ and placed in a previously cleaned beaker for the lift-off procedure, protein coating and cell culturing. The lift-off and cell culturing procedures are described in the Supplementary Material.