Mixed hippocampal neuroglial cultures and pure glia cultures were prepared from P2 rats as previously described (21). Neurons (between 10 and 14 DIVs) were transfected with the calcium-phosphate ProFection transfection kit (Promega, Fitchburg, WI, USA) as previously described (21). The use of this kit increased the reproducibility over time. Briefly, cells were incubated for 30 min in 37°C fresh Dulbecco’s modified Eagle’s medium (DMEM complemented with 10 mM MgCl2 and 5 mM Hepes at pH 7.5). For each coverslip, 50 μl of transfection mixture containing 2.5 μg of plasmid DNA, 200 mM CaCl2, and 1× Hepes-buffered solution was applied. The cultured neurons were incubated with the transfection mixture for 20 min at 37°C and 5% CO2 followed by three washes with fresh DMEM. Coverslips were placed back into their original culture medium and were cultured for 3 days at 37°C and 5% CO2 to allow transgene expression or down-regulation of the targets before further analyses. Human embryonic kidney (HEK) 293T, C6, and PC-12 cell lines were purchased from the German National Resource Center for Biological Material (DSMZ) and cultured following the recommended conditions. For transfection, PC-12 and C6 cells were detached by trypsin treatment and transfected using the SF cell line 4D-Nucleofector Kit (Lonza) following the instructions of the manufacturer. Following electroporation, transfected cells were seeded and incubated for 3 days at 37°C. The efficiency of down-regulation through TDP-43 antisense (TDP-43-AS) was tested in C6 cells followed by qRT-PCR for TDP-43. Differentiation of PC-12 cells was induced 1 day after transfection with plates coated with collagen (2 μg/ml; Roche) by adding 0.1% horse serum and nerve growth factor RPMI medium (50 ng/ml). To induce differentiation, PC-12 cells were cultured in this medium for 7 days, and the medium was changed every second day. Differentiation was assessed by morphological changes (neurite formation).

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