Edta 0.02 solution

Two gastric adenocarcinoma cell lines were used: the EPG85-257 P – sensitive and EPG85-257 RDB – daunorubicin-resistant cell line. Human gastric cancer cells were obtained as a kind gift from Prof. Herman Lage. The EPG85-257 RDB cell line reveals the overexpression of MDR1 and the resistance to daunorubicin [37 (link)]. Both cell lines were cultured in Leibovitz L-15 medium (Sigma-Aldrich, Poznan, Poland) supplemented by 10% fetal bovine serum (FBS, Lonza, Poland), 1% antibiotics (penicillin/streptomycin, Sigma-Aldrich, Poznan, Poland), 1 mM ultraglutamine (Sigma-Aldrich, Poznan, Poland), 6.25 mg/L fetuin (Sigma-Aldrich, Poznan, Poland), 2.5 mg/mL transferrin (Sigma-Aldrich, Poznan, Poland), 0.5 g/L glucose, 1.1 g/L NaHCO₃, and 1% minimal essential vitamins (Mem-Vit, Sigma-Aldrich, Poznan, Poland). Cell cultures were cultured as a monolayer on a 25 and 75 cm² plastic flask (Sarstedt, Germany) maintained in a humidified atmosphere at 37 °C and 5% CO₂, and detached for the experiments by trypsinization (trypsin 0.025% and EDTA 0.02% solution, Sigma-Aldrich, Poznan, Poland). Cells were passed every 2–3 days and a day before the experiment.

The studies were performed in vitro on human breast adenocarcinoma cell line doxorubicin-resistant type (MCF-7/DX), obtained from the Department of Tumor Biology, Comprehensive Cancer Center, Maria Sklodowska-Curie Memorial Institute in Gliwice (Poland), human amelanotic melanoma cells (C32) purchased in ATCC^®, MC38 murine colon adenocarcinoma cells were adapted to in vitro conditions in Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (Wroclaw, Poland)^{33 (link)}. MCF-7/DX and C32 cells were grown in DMEM (Sigma, Poland), supplemented with 10% fetal bovine serum (Lonza BioWhittaker, Switzerland) and penicillin/streptomycin (Sigma, Poland). MC38 cells were maintained RPMI (Sigma, Poland) supplemented with 5% fetal bovine serum (FBS, Lonza BioWhittaker, Switzerland), 1% penicillin/streptomycin (Sigma, Poland), 0.5% sodium pyruvate (Sigma-Aldrich) and 50 µmol/L 2-mercaptoethanol (Sigma-Aldrich). Cell cultures were cultivated as a monolayer on a plastic flask 25 and 75 cm² (Nunc, Denmark), maintained in a humidified atmosphere at 37 °C and 5% CO₂. and detached for the experiments by trypsinization (trypsin 0.025% and EDTA 0.02% solution, Sigma, Poland). Cells were passed every 2–3 days and a day before the experiment.