Hyclone 10 fetal bovine serum fbs

A 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used in order to investigate the ability of cetuximab to inhibit the proliferation of the 3 OSCC lines (OSC-19, OSC-20 and HOC313) in vitro. Initially, 2,500 cells/well were cultured in Dulbecco's modified Eagle's medium (DMEM; D7440; Sigma-Aldrich Japan K.K.) supplemented with Hyclone™ 10% fetal bovine serum (FBS; GE Healthcare, Logan, UT, USA), in 96-well tissue culture plates. Following 24 h of culture, the cells were treated with varying concentrations of cetuximab (Erbitux®; Merck Serono Co., Ltd., Tokyo, Japan; 0, 0.005 and 0.01 µg/ml) diluted in DMEM supplemented with 2% FBS. In order to measure the number of metabolically active cells following 24, 48 and 72 h of incubation, an MTT assay was performed and the results were quantified according to the manufacturer's protocols. The optical density at 450 nm was measured using a microplate reader (iMark Microplate Absorbance Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A375 cells were purchased from the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). B16F0 cells were obtained from the China Center for Type Culture Collection (Wuhan, China). SK-MEL-5 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). A375 cells were cultured in Gibco™ DMEM, while SK-MEL-5 and B16F0 cells were cultured in Gibco™ RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing HyClone™ 10% fetal bovine serum (FBS; GE Healthcare Life Sciences), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cell lines were cultured at 37°C in 5% CO₂. Cells were allowed to attach for 24 h before treatment. LD was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and diluted with fresh medium to achieve the desired concentration. The final concentration of DMSO did not exceed 0.2% in the fresh medium, and DMSO at this concentration has no significant effect on cell viability.