Generation of Tumor-Conditioned Media

Supernatant obtained from B16F10 cells (Korean Cell Line Bank) was used to mimic the tumor microenvironment [3 (link)]. B16F10 cells were seeded and cultured with complete DMEM in a 100-mm cell culture dish at 37°C and 5% CO₂ until confluency (~90%). The supernatant was collected and then filtered through a 0.2-μm filter. The filtered-supernatant was aliquoted and stored at -20°C until used. We named the B16F10-conditioned media “100% TCM” which was regarded as glucose-depleted because these cells potentially consumed nearly all the glucose in complete DMEM while growing. Two types of TCM mixture (% of TCM) composed of B16F10-conditioned media and DMEM were used; one was “glucose-containing TCM mixture” that consisted of 100% TCM and 25 mM glucose-containing complete DMEM, and the other was a “glucose-depleted TCM mixture” that was composed of 100% TCM and glucose-free DMEM.