Axio version 4

OVCAR-8 spheroids were created using the hanging drop method as previously described (25 (link)). A total of 15 µl of the medium that contained 5×10³ cells were added to each circle on the inverted cover of a 96-well plate to create one spheroid. The cover was then placed upside down on the plate coated with sterile agarose 1.5% (w/v) containing 200 µl complete medium. Following 48 h of incubation in a humidified chamber with 5% CO₂ at 37°C, spheroids were transferred from the cover into each well of the agarose-coated plate and further cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). Spheroids were treated with oxostephanine under two conditions: i) The compound was added to the cell preparation before making the hanging drop; and ii) the compound was added after transferring the formed spheroids into the culture wells. Two concentrations at 5 and 1 µM of Oxostephanine were used in both conditions. Images were obtained using an Axiovert 40CFL microscope (Carl Zeiss AG) with Powershot G9 camera. These images were analyzed using Axio version 4.5 software (Carl Zeiss AG) to determine the spheroid diameter. The approximated volume (V) of each spheroid was calculated as follows: V= (4/3) x π x (D1/2) x (D2/2)², where D1 and D2 were the longest and shortest diameters, respectively (26 (link)).