Cell Culture and Transfection Protocol

The plasmid expressing the surrogate reporter gene, pMRS-CMV (Toolgen) was transfected to HEK293T cells using Lipofectamine 3000 according to the manufacturer’s instructions. RFP-positive cells were sorted using Moflo Astrios EQ (Beckman Coulter), followed by selection and culture of cells with the highest RFP signals. CT26 murine colon cancer cells, 4T1 murine breast cancer cells, RENCA murine kidney cancer cells, Raw264.7 murine macrophage cells, and HEK293T human embryonic kidney cells were obtained from the American Type Culture Collection (ATCC). DCON murine gastric cancer cells were kindly provided by Dr. Sam S. Yoon (Memorial Sloan Kettering Cancer Center) and Dr. Sandra Ryeom (University of Pennsylvania) [42 (link)]. CT26, 4T1, and RENCA cells were cultured in RPMI-1640 (Gibco) supplemented with L-glutamine, 25 mM HEPES (Gibco), 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Gibco). DCON, Raw264.7, HEK293T cells were cultured in DMEM (Gibco) containing the same supplements as RPMI 1640. Cells was cultured in a CO₂ incubator (BB15, Thermo Fisher) at 37 ℃ at 5.0% CO₂ condition.