Zen core v3

Adrenocortical carcinoma MCTS were generated by the liquid overlay method [26 (link)]. Briefly, 50 µL of an autoclaved and thereby liquefied 1.5% (w/v) agarose-PBS solution was aseptically added to each well of a 96-well microtiter plate. After cooling down for at least 30 min, 8000 cells per well were seeded in a volume of 200 µL. The plates were then centrifuged at 600× g and 20 °C for 5 min (Centrifuge 5804 R, Eppendorf AG, Hamburg, Germany) and incubated for 4 days until reaching a spheroid diameter of approximately 475 µm and circularity of 0.9. Circularity was calculated using ImageJ software as previously described [32 (link)]. Spheroid formation and growth were monitored using an Axiovert 40 CFL microscope with an Axiocam 305 color camera and a ZEN core v3.0 software (Carl Zeiss, Oberkochen, Germany).

LS spheroids were cultured in RPMI 1640 medium w/o phenol red and treated with non-PEGylated or PEGylated Cu(DDC)₂ liposomes. Spheroids were stained with 1 µM calcein-AM and 5 µM PI for the last 2 h of a 72 h incubation period of Cu(DDC)₂ liposomes. Living and dead cells were analyzed with an Axiocam 305 color camera, a ZEN core v3.0 software (Carl Zeiss, Oberkochen, Germany), a 10× objective and a corresponding filter set for calcein-AM and propidium iodide.