Model bh2 rfl t3

To visualize apoptotic cell death and nuclear morphology, SK-MES-1 cells were stained with Hoechst. Briefly, SK-MES-1 cells were seeded into 6-well flat bottom plate at density of 6×10⁵cells/well and treated with Isoalantolactone at the concentration of 0, 20, 40 μM respectively. After 24 h of treatment, cells were collected, washed and allowed to dry on slides. Then nuclei were stained with Hoechst for 10 min. The apoptotic cells displaying fragmented or condensed nuclei were observed under a fluorescence microscope (Model:BH2-RFL-T3, Olympus, Tokyo, Japan).

Spheroids were washed with 100 µL phosphate buffered saline (PBS), and afterwards incubated with propidium iodide (Sigma-Aldrich, St. Louis, USA; 1:500 in medium) for 5 h 45 min followed by the addition of calcein acetoxymethyl ester (Sigma-Aldrich; 4 mM in dimethyl sulfoxide, 1:800 in medium) with further incubation for 15 min. Spheroids were then transferred to flat-bottom 96-well plates, washed with PBS and imaged under an IMT-2 Inverted fluorescence Microscope (Olympus) equipped with a 100 W Mercury Power Supply (Model BH2-RFL-T3, Olympus) for fluorescence detection. Diameters of total spheroid and necrotic core were measured using the Digimizer software version 5.7.2 (MedCalc Software Ltd, Ostend, Belgium).