D mem medium high glucose

B16 mouse melanoma cells [35] (link) were transfected with the pcDNA3-mHEL plasmid by lipofection using Trans IT-LT1 (Takara), and cultured with G418 (2 mg/ml, Wako). Drug-resistant stable clones (B16-mHEL) were subsequently selected. B16 cells were retrovirally transduced with the pMX-mHEL-IRES-GFP, and then cloned by limiting dilution method to establish B16-mHEL-GFP cells. 40 LB, Balb/c 3T3 fibroblasts expressing exogenous CD40-ligand and BAFF, have been described previously [17] (link). 40 LB cells were transduced with the pMX-mHEL-IRES-GFP vector, and a single clone expressing mHEL and eGFP, termed 40 LB-mHEL, was selected by limiting dilution. To express FasL, 40 LB cells were first transduced with the pSIREN-RetroQ-shFas vector. The resultant Fas-knocked-down cells (40 LB-Fas⁻) were then transduced with the pMX-FasL-IRES-hCD8 vector and a single clone expressing FasL and hCD8 (40 LB-FasL cells) was selected by limiting dilution. Finally, the 40 LB-FasL cells were transduced with the pMX-mHEL-IRES-GFP vector to obtain a single clone expressing mHEL and eGFP (40 LB-mHEL-FasL). B16 and 40 LB cells, and their derivatives, were maintained in D-MEM medium (high glucose; Wako) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (GIBCO) in a humidified atmosphere at 37°C with 5% CO₂.

B16-mHEL-GFP mouse melanoma cells [13 (link)] and 40LB [11 (link)] were maintained in D-MEM medium (high glucose; Wako) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (GIBCO) in a humidified atmosphere at 37°C with 5% CO₂.