Hoechst 33342 hoechst

HepG2 cells in 75 mL culture flasks were trypsinized with 3 mL of 0.05% trypsin solution (Sigma, WI, USA), and incubated for approximately 5 min at 37 °C. The trypsin was neutralized by addition of complete culture medium, before the cultures were centrifuged at 1 000 r/min (radius = 98 mm) for 5 min. Cells were harvested and resuspended in medium, to ensure a single cell suspension. Cells were then counted on a hemocytometer based on the trypan blue dye exclusion method (0.4% trypan blue; Sigma-Aldrich, St Louis, MO, USA) by adding 10 μL of trypan blue solution to 10 μL of cell suspension (1:1, v/v). The cells were seeded at densities ranging from 500 to 6 000 cells per well in four replicates, using black, tissue culture treated, clear-bottom 384-multiwell plates. Cells were incubated for 24 h prior to treatment with varying concentrations (0.062 5%, 0.125%, 0.25%, 0.5% and 1.0%) of dimethyl sulfoxide (DMSO) solutions prepared with the complete medium. Cell cultures were then incubated for 24 h before staining with Hoechst 33342 (Hoechst) (Invitrogen, Eugene, OR, USA) and propidium iodide (PI) (Sigma, MO, USA), prepared in culture medium, at final concentrations of 5 and 10 μmol/L respectively for 1 h. Plates were imaged with an ImageXpress MicroXL high-content fluorescence microscope (Molecular Devices, Sunnyvale, CA, USA).