Amphotericin b as fungizone

Human gastric carcinoma cell line GTL‐16 was provided by P. Comoglio (Medical School University of Torino, Italy), nonsmall cell lung cancer cell line EBC‐1 by S. Giordano (University of Torino, Torino, Italy), and pharyngeal carcinoma line Detroit 562 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). GTL‐16 and EBC‐1 cells were cultured in RPMI (Gibco, Invitrogen, Reinach, Switzerland) supplemented with 5% and 10% FCS (Sigma‐Aldrich Inc., St. Louis, Missouri, USA), respectively, and an antibiotic/antimycotic solution (penicillin 100U·mL⁻¹, streptomycin sulfate 100U·mL⁻¹, amphotericin B as fungizone 0.25µg·mL⁻¹; Gibco). Detroit 562 cells were maintained in MEM (Sigma‐Aldrich) supplemented with 10% FCS, nonessential amino acid solution (1% vol./vol.; Sigma‐Aldrich), and antibiotic/antimycotic solution.

The EoL-1 cell line was provided by Genotype SA (Athens, Greece). The cells were cultured in RPMI 1640 medium (Biochrome KG, GIBCO 31870, Co Dublin, Ireland) supplemented with 10% heat-inactivated fetal bovine serum (Fetal Bovine Serum-FBS, 10270, GIBCO, Co Dublin, Ireland), 2 mM glutamine (L-Glutamine 200 mM, 25030, GIBCO, Co Dublin, Ireland), penicillin 100 units/mL (Penicillin G sodium 10,000 units/mL, GIBCO, Co Dublin, Ireland) and streptomycin 100 mg/mL (Streptomycin Sulfate 10,000 μg/mL and 25 μg/mL amphotericin B as fungizone, GIBCO, Co Dublin, Ireland) at 37 °C in a humidified atmosphere of 5% carbon dioxide (SANYO CO₂, INCUBATOR, LabX, Midland, ON, Canada). The above complete medium was renewed every 3–4 days.
The cell number was adjusted to 1 × 10⁶ cells/mL with fresh complete medium. The viability of the cells was measured by Trypan Blue exclusion test (Blue solution 0.4% in NaCl, SIGMA, 0.2% final in PBS, phosphate buffer saline, GIBCO, Co Dublin, Ireland) every 3 days by cell counting on a Neubauer hemocytometer with a microscope (Kruss, Hamburg, Germany), as well as by MTT assay (CellTiter 96^® AQueous One Solution Cell Proliferation Assay, Promega Corporation, Madison, WI, USA).