The effect of Piptoporus betulinus extract and the saturated ethanolic solution of betulin on Hs27, WM115, and A375 cells was tested by cell viability assays: colorimetric CellTiter (G4000, Promega, Madison, WI, USA) and fluorometric alamarBlue (DAL1025, Thermo Fisher, Waltham, MA, USA), cytotoxic lactate dehydrogenase (LDH) activity test (MAK066, Sigma-Aldrich, St. Louis, MO, USA), as well as ApoTox-Glo assay (G6320, Promega, Madison, WI, USA) were used to analyze both cell viability and apoptosis.
Cells, detached by trypsinization and counted after trypan blue staining (Bio-Rad, TC-10, Hercules, CA, USA), were seeded in 96-well flat-bottomed plates (1–2 × 104 cells per well), either transparent (TPP) or black (Invitrogen, Paisley, UK), and incubated overnight. Cells in each plate were treated for 24 h with different concentrations of Piptoporus betulinus ethanolic extract (1, 2.5, 5, and 10 μL mL−1), the saturated ethanolic betulin solution (0.01, 0.05, 0.25, 1, 5, 7.5, and 10 μL mL−1), or the highest concentration (10 μL mL−1) of ethanol (EtOH) (Figure 7 ).
Cells, detached by trypsinization and counted after trypan blue staining (Bio-Rad, TC-10, Hercules, CA, USA), were seeded in 96-well flat-bottomed plates (1–2 × 104 cells per well), either transparent (TPP) or black (Invitrogen, Paisley, UK), and incubated overnight. Cells in each plate were treated for 24 h with different concentrations of Piptoporus betulinus ethanolic extract (1, 2.5, 5, and 10 μL mL−1), the saturated ethanolic betulin solution (0.01, 0.05, 0.25, 1, 5, 7.5, and 10 μL mL−1), or the highest concentration (10 μL mL−1) of ethanol (EtOH) (