**2.14. Cell morphology evaluation by inverted light microscopy**

Hep-2 cell cultures (2× 105 cells/mL) were prepared in 96-well tissue culture plates (Greiner-Bio one, Germany). After 24 h incubation at 37 °C in a humidified 5% (v/v) CO2 atmosphere cell monolayers were confluent, the medium was removed from each well and replenished with 100 \_L of bi-fold dilutions of different samples tested prepared in DMEM (GIBCO BRL). For cell controls 100 \_L of DMEM without samples was added. All cultures were incubated at 37 °C in a humidified 5% (v/v) CO2 atmosphere for 72 h. Cell morphology was observed daily for microscopically detectable morphological alterations, such as loss of confluence, cell rounding and shrinking, and cytoplasm granulation and vacuolization. Morphological changes were scored (Simoes et al., 1999).
