Rpmi 1640 medium hepes modification

Horse serum was purchased from Biochrom AG (Berlin, Germany). The protein-assay dye reagent concentrate was from Bio-Rad (Munich, Germany) and the GelGreen nucleic acid gel stain was from Biotium (Hayward, CA, USA). Hematopoietic Growth Medium (HPGM) was from Lonza (Cologne, Germany). Recombinant human fms-related tyrosine kinase 3 ligand (Flt3), purified recombinant human stem cell factor (SCF) and recombinant human thrombopoietin (TPO) were from MACS Miltenyi Biotec (Gladbach, Germany). Mitomycin C was from Medac (Hamburg, Germany) and methanol was from Carl Roth (Karlsruhe, Germany). 1,4-Diazabicyclo[2.2.2]octane (DABCO), albumin from human serum, cytochalasin B, L-glutamine solution, methyl methanesulfonate, penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin per ml), RPMI 1640 medium (HEPES modification) and sodium pyruvate solution were purchased from Sigma-Aldrich (Munich, Germany). Doxorubicin hydrochloride, Mitomycin C and vinblastine sulfate were from Teva (Ulm, Germany).

The human NSCLC cell line HCC827 (American Type Culture Collection Co., MD, USA) was exposed to 1 μM of gefitinib for 48 h in RPMI-1640 Medium HEPES Modification (Sigma-Aldrich Co., MO, USA) containing 10% fetal bovine serum (FBS; Invitrogen^™ Thermo Fisher Scientific Inc., MA, USA) and 1% penicillin-streptomycin (P/S; Invitrogen^™ Thermo Fisher Scientific Inc.). The cells were then washed and cultured in drug-free medium until surviving cells were 80% confluent. These cells were then re-exposed to increasing concentrations of gefitinib, from 1 to 5 μM. Cells that were finally able to grow in 5 μM gefitinib were obtained 1.5 months after the initial exposure as described previously [12 (link)]. The established resistant cells were maintained in medium containing 1 μM of gefitinib. For all in vitro studies, resistant cells were eventually cultured in drug-free medium for at least 1 week to eliminate gefitinib. All cells were maintained under a humidified atmosphere of 5% CO₂ at 37°C. Gefitinib-resistant HCC827 cells are referred to here as HCC827GR cells.