Mithras2 lb 943 multimode reader

We evaluated cell viability to make sure that the results obtained for DAO activity were not biased by the sensitivity to different cell lines to transfection. The viability of transfected cells was determined using CellTiter 96^® AQueous One Solution Cell Proliferation Assay (G3582, Promega) according to manufacturer’s guidelines. HEK293, 1321N1 and SH-SY5Y cells were seeded into 96-well plates (83.3925, Sarstedt) at a density of 2 × 10⁴ cells, 5 × 10⁴ and 1 × 10⁵ cells in 100 μL growth medium, respectively. They were allowed to adhere for 24 h and were transfected with 1.2 μg of plasmids using Xfect transfection reagent (631317, Takara Clontech) according to manufacturer’s guidelines. The growth medium was exchanged 4 h after transfection. Each transfection condition was analyzed in triplicates. The cells were incubated for 48 h following transfection. Then, 20 μl of CellTiter 96^® AQueous One Solution Reagent was added to each well and incubated at 37°C for 4 h. The absorbance was measured at 490 nm using Mithras² LB 943 Multimode Reader (Berthold Technologies). The absorbance values were corrected by subtracting the average absorbance from the control wells with no cells.