The cell cycle distribution was analyzed using a FACSCaliburTM (BD Biosciences, Heidelberg, Germany), as reported [20 (link)]. Briefly, cells were harvested, washed with PBS, fixed in chilled 70% ethanol at 4 °C for 30 min., treated with 1 mg/mL of RNase A (Sigma-Aldrich, Munich, Germany) and stained with 100 μg/mL of propidium iodide (PI) for 30 min. at 37 °C. DNA content was determined.
Cell proliferation assays were carried out by using Cell Titer-Blue® Cell Viability Assay (BD Biosciences, Heidelberg, Germany) on treated cells in 96-well plates (Promega, Mannheim, Germany). 20 μL of CellTiter-Blue® reagent was added to each well and then incubated at 37 °C with 5% CO2 for 4 h before fluorescence reading while using a Victor 1420 Multilabel Counter (Wallac, Finland), as reported [20 (link)].
Cell proliferation assays were carried out by using Cell Titer-Blue® Cell Viability Assay (BD Biosciences, Heidelberg, Germany) on treated cells in 96-well plates (Promega, Mannheim, Germany). 20 μL of CellTiter-Blue® reagent was added to each well and then incubated at 37 °C with 5% CO2 for 4 h before fluorescence reading while using a Victor 1420 Multilabel Counter (Wallac, Finland), as reported [20 (link)].
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