Spheroid formation
For the generation of tumor spheroids in 3D, 200 μL/well of cell
suspension in culture medium was seeded at cell densities of 20,000
cells/well. NPC (C17/NPC43+ve) cells were dispensed into Nunclon Sphera
96 wells plate (Thermo Fisher Scientific). The Nunclon Sphera surface
was designed to cause minimal cell attachment with minimal extracellular
matrix protein binding to the plate surfaces. Plates were incubated at
37 °C and 5 % CO2 in RPMI supplemented with FBS and
ROCK inhibitor. To determine the IC50 values, spheroids
were stained with 0.1 uM Invitrogen Cell Event (diluted by PBS + 5%
FBS) and 0.2 uM Lysotracker in 100 uL Gibco Fluorobrite DMEM with 10 %
FBS in the presence of indicated doses of IDX and cis.