Xcelligence real time cell analyzer system

Cell proliferation and adhesion were monitored using the xCELLigence Real-Time Cell Analyzer (RTCA) system as per manufacturer’s instruction (Roche Applied Science, Mannheim, Germany). Cells (10,000 cell/well/100 µl) were seeded on E-Plate 16, allowed to attach onto the electrode surface, and then replaced with fresh medium containing SR, VF, or SRVF either from the beginning or 20 h post-seeding. The electrical impedance was automatically recorded every 15 min and expressed as a Cell Index (CI) value.

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).

To analyze the effect of GARP on ASC proliferation, the xCelligence real‐time cell analyzer system from Roche (Roche Applied System, Penzberg, Germany) was used. In brief, 1000 cells/well of NT, LV‐CTRL, LV#18, LV#19, or LV#19 + LV‐GARP ASCs were added to 16‐well E‐plates (Roche Applied System) as previously described.43 The E‐plates were then placed on the device station in the incubator (21% O₂/5% CO₂ at 37°C) for the continuous recording of impedance, as reflected by cell index. In order to understand the mechanisms behind the inhibition of proliferation in GARP^−/lowASCs, NT, LV‐CTRL, LV#18, and LV#19 ASCs were treated with SB431542 (10 μM), N‐acetyl cystein (NAC, 1 mM), apocynin (5 mM), mitoTEMPO (25 μM) (all inhibitors are from Sigma‐Aldrich) or anti‐TGF‐β1/2/3 Ab (11D1, R&D Systems, Minneapolis, Minnesota), starting 1 day after GARP silencing. Cells were harvested 3 days later and added to 16‐well E‐plates (1000 cells/well), and the proliferation was followed as described above. Fresh media with or without inhibitors were added every 3 days. To analyze the proliferation of NT(S) and GARP⁺⁺ASCs, cells were plated at low density (50 000 cells/well) in 6‐well plates. When the cell cultures reached an 80‐90% of confluence, cells were harvested, counted, and replated at the same concentration. The proliferation was followed for 3‐4 weeks.

Cell proliferation assays were performed using the xCELLigence Real-Time Cell Analyzer system (Roche, Switzerland). Cells were seeded in a 16-well E-plate at 30% confluence with 200 μl medium/well, and two replicates on an E-plate were performed. Cytotoxicity experiments were performed by adding ribociclib (MedChemExpress, USA) to the system 14 h after the cells were seeded into the E-plate. Proliferation and drug toxicity assays were repeated 3 times using MTS experiments (Promega, USA) according to the protocol.