Las af frap application wizard

NSC-34 cells were plated in eight-well plates (density, 22,500 cells per well) and transfected with 25 μg of wtTDP43tdTOMATOHA plasmid (28205, Addgene) using 1.1 μl of Lipofectamine 3000 Reagent, 1.5 μl of transferrin (5 mg/liter), and 0.75 μl of P3000 reagent in DMEM for 3 hours in a 5% CO₂ humidified atmosphere at 37°C. After 3 hours, the DMEM was replaced with fresh complete cell medium, and the FRAP experiment was performed after 40 hours using the LAS AF FRAP Application Wizard on the Leica TCS SP8 3× microscope. Images were acquired using the Leica HC PL APO CS2 100×/1.40 oil white objective and the 554-nm laser, at a scan speed of 700 Hz. A prebleach image was acquired with a laser power of 20%. Photobleaching was then performed for 10 s in ROIs containing TDP-43 inclusions, at 60 to 80% laser intensity. Fluorescence recovery was then monitored for 20 to 30 s by setting the laser power back to 20%.