Real-time Cell Proliferation Assay for ASCs

Proliferation of ASCs was measured using the xCelligence real-time cell analyzer system from Roche (Roche Applied Science, Penzberg, Germany, http://www.roche.com). In brief, 800 cells/well of NT, LV-CTRL, LV#3, and LV#6 mASCs or NT, LV-CTRL, LV#18, and LV#19 hASCs were added to 16-well E-plates as described previously 30 (link). The E-plates were then placed on the device station in the incubator (normoxia; 5% CO₂ at 37°C) for continuous recording of impedance, as reflected by cell index. In some experiments, a TGF-β1/2/3-neutralizing antibody (1D11; R&D Systems, Minneapolis, MN, http://www.rndsystems.com) or SB431542 (Sigma) was added on days 1 and 3 to the wells at 2.5 µg/ml and 10 µM, respectively. Cell proliferation was also assayed using the CellTiter-Blue reagent, according to the manufacturer's instructions (Promega, Madison, WI, http://www.promega.com). In brief, NT, LV-CTRL, LV#3, and LV#6 mASCs were seeded in 96-well plates (800 cells/well) and cultured at 5% O₂/5% CO₂ at 37°C. On days 1, 3, and 7, CellTiter-Blue was added to the cells during the last 4 hours of culture and fluorescence was measured at 560 nm on a Glomax multidetection system (Promega).