2.4. Cell Viability

Cells were seeded in 96-well plates at the concentration of 3 × 10³ cells/well. After 48 h, cells were serum starved for 12 h and further incubated with the different stimuli for a 24 h, 48 h, or 72 h. Cell viability was assessed by MTT assay, as reported previously [16]. Briefly, cells were incubated with 1 mg/mL of MTT for approximately 2 h, then the medium was removed, and formazan products solubilized with 100 μL DMSO. Cell viability was assessed by spectrophotometry at 570 nm absorbance using the LT-4000 microplate reader (Euroclone, Milan, Italy). In experiments using human primary PA cells, 1 × 10⁴ cells/well were plated in 96-well plates to assess the effect of the different stimuli on cell viability every 24 h until 72 h using Alamar blue reagent (Invitrogen, Madrid, Spain), as described previously [34,35]. Treatments were daily refreshed after each measurement, and cell viability was evaluated using Flex-Station III System (Molecular Devices, Sunnyvale, CA, USA).