Culturing and Characterizing Mouse Cancer Cell Lines

The LLC cells used in this study were the LLC-Mhi cell line (obtained from Dr. Glenn Merlino, NCI), which is a high metastatic subline derived from LLC tumors described previously (Day et al., 2012 (link)). B16F10 cells were purchased from American Type Culture Collection. ARH-77 and ARH-77-mSDC1 cells were kindly provided by Dr. Ralph D. Sanderson (University of Alabama at Birmingham), and RAW264.7 cells by Dr. Raymond P. Donnelly (FDA). LLC, ARH-77, and RAW264.7 cells were all tested negative for mycoplasma (NCI Core facility) and authenticated by STR analysis (IDEXX BioResearch). LLC and B16F10 cells were cultured in RPMI 1640 Medium (LONZA) with heat-inactivated fetal bovine serum (FBS), supplemented with penicillin/streptomycin (1:100) at 37 ˚C, 5% CO₂. Culture of LLC cells was carried out under various concentrations of FBS, as indicated in the Figure legends. For LPS stimulation, RAW264.7 cells were cultured in OPTI-MEM^TM I reduced serum medium (Thermo Fisher Scientific) for times indicated in the text. LPS transfection was performed using X-tremeGene HP DNA transfection reagent (Roche Applied Science).