Donepezil

Before the experiments were started we determined the most effective concentration (EC) of AChE inhibition by donepezil (Sigma-Aldrich, Taufkirchen, Germany) using the AChE Assay Kit, a sequential series of donepezil (5 µM, 1 µM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM), and 4,800 MSC per well (96 well plate). After donepezil treatment for 24 h, cells were homogenized and 50 µL of the supernatant was incubated with 50 µL AChE assay mixture according to the protocol of the manufacturer (see above). After incubation in the darkness, samples were measured and the EC values were determined because of the direct relation of measured fluorescence and AChE activity (Figure 1A, EC30 11.1, EC50 24.6, EC70 54.7). The experiment was repeated with a 1:2 dilution of the cells to verify the reproducibility and linearity of the values (Figure 1B, EC30 19.7, EC50 38, EC70 73.5). The means were calculated as final EC values: EC30 15.4, EC50 31.3, EC70 64.1. To determine which EC value was most suitable to stimulate proliferation the BrdU assay was conducted as described above (Figure 1C). Since the highest value could be measured after using EC30 we decided to use 15.4 nM donepezil for the following experiments.