Transient transfection of hMSC-TERT4 with reporter plasmids and luciferase assay. hMSC-TERT4
VER155008 were seeded on pre-coated silicone dishes and cultured for up to 2 days in MEM supplemented with 10% FCS. For transfection, culture medium was changed and the cells were treated with plasmid DNA (2 μg), pSV-β-galactosidase control vector DNA (2 μg) and 6 μl Fugene 6 transfection reagent (Roche Diagnostics, Mannheim, Germany) in 94 μl serum-free MEM. The transfected cells were further cultured for 48 h. Thereafter the silicone dishes with transfected cells were placed in the mechanical stimulation apparatus and stretched homogenously at 50,000 με at a frequency of 1 Hz for a total of 5400 cycles (equivalent to 1.5 h). Transfected control
cells were treated in parallel without loading. After loading, the cells were washed with PBS and were scraped in 400 μl 1x reporter lysis buffer (Promega). Luciferase activities in the cell lysates were determined using the Centro LB 960 microplate luminometer (Berthold Technologies, Bad Wildbad, Germany) and the luciferase assay system (Promega), according to the manufacturer’s instructions. β-Galactosidase activity was measured with the β-galactosidase enzyme assay system with reporter lysis buffer (Promega) according to the manufacturer’s instructions. Luciferase activity of each sample was normalized to that of β-galactosidase.
