Cell Viability, Proliferation, and Cycle Analysis

Cell viability was determined using MTT metabolism test, as described [20 (link),33 (link)]. Cell proliferation was assessed using ELISA BrdU kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. For cell cycle analysis, cells were collected by trypsinization, fixed in 70% ethanol and stained with propidium iodide solution (3.8 mM sodium citrate, 50 mg/ml RNAse A, 500 mg/mL PI, in PBS). DNA content analyses were performed using FACS Calibur flow cytometer (BD Biosciences, Waltham, MA, USA) and the BD CellQuest Pro 6.0 software (BD Biosciences, San Jose, CA, USA). At least 10,000 events were analysed for each sample.