Dmem with 4.5 g l glucose

Peripheral blood mononuclear cells (PBMCs) were prepared from leukopaks obtained from healthy donors from LifeSouth Community Blood Center (Gainesville, FL) under approval by the Institutional Review Board at the University of Florida. PBMCs were isolated by centrifugation on LymphoSep® Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA) medium. Monocytes were isolated by positive selection on LS Columns (Miltenyi Biotec, Bergisch Gladbach, Germany) with human CD14 MicroBeads (Miltenyi Biotec). Freshly isolated monocytes were differentiated into macrophages in MDM medium (DMEM with 4.5 g/L glucose (Corning, Corning, NY), 10% heat-inactivated human serum (Zen-Bio, Research Triangle Park, NC), 2 mM L-glutamine (Corning), and 100 IU penicillin-streptomycin (Corning)) supplemented with 10 ng/ml M-CSF for 7–10 days. After differentiation, the medium was replenished without M-CSF and the cells rested overnight before being used for experiments.

CMT167 cells were stably transfected with firefly luciferase, as previously described (24 (link)). Luciferase-expressing LLC cells were purchased from Caliper Life Sciences (LL/2-luc-M38). The CMT167 cells harbor a Kras^G12V mutation, and the LLC cells harbor a Kras^G12C mutation (24 (link)). Both cell lines were maintained in DMEM with 4.5 g/l glucose (no. 10–017-CV; Corning) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml G418 at 37°C in a humidified 5% CO₂ atmosphere. Cells were periodically tested for mycoplasma infection, maintained as frozen stocks, and cultured for only 2–4 wk before use in experiments. Authentication of cell lines based on morphology, growth curve analysis, and metastatic phenotype was performed regularly. Human NSCLC cell lines were maintained in RPMI 1640 (no. 10–040-CV; Corning) containing 10% FBS at 37°C in a humidified 5% CO₂ atmosphere. All were KRAS mutant.