75 cm2 culture flask

Mucus-secreting HT29-MTX cells (Sigma–Aldrich) were routinely grown in 75 cm² culture flasks (Sarstedt, Nümbrecht, Germany), under a humidified atmosphere containing 5% CO₂, at 37°C, in DMEM (PAN Biotech) supplemented with penicillin/streptomycin (100 U/mL), 2 mM L-glutamine, and 10% heat-inactivated fetal bovine serum.

Human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells were purchased from DSMZ (German collection of microorganisms and cell cultures, Braunschweig, Germany). CCRF-CEM and HEK293 cells were grown in 75-cm² culture flasks (Sarstedt, Nuemberecht, Germany) and maintained in culture at 37 °C in 95% humidity, 20% O₂ and 5% CO₂. RPMI-1640 and DMEM culture medium supplemented with 10% fetal calf serum, 100,000 U/L penicillin and 100 μg/L streptomycin (Biochrome, Berlin, Germany) were used to grow CCRF-CEM and HEK293 cells, respectively. Cell confluency was regularly checked, and the medium was changed accordingly.

AGS cells (human gastric epithelial cell line) were obtained from the American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) (Lonza, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare, Logan, UK) and 1% penicillin/streptomycin (5000 U/mL) (Lonza) was used to culture the cells. Around 1 × 10⁶ cells were plated in 75 cm² culture flasks (Sarstedt, Barcelona, Spain) and maintained at 37 °C until 90% of cell confluence under 5% CO₂ in a humidified incubator. Cell culture medium was changed every 2 days. Cell subculturing was carried out before a confluent monolayer appeared. Experiments were developed between passage 10 to passage 30 to ensure cell uniformity and reproducibility.

The cancer cell lines in this study, MCF-7, A427, RT-4, SiSo, LCLC-103H, DAN-G, KYSE-70, A2780, HL-60, and U-937 were obtained from the German Collection of Microorganisms and Cell Culture (DMSZ Braunschweig, Germany) (Table 4). The A2780 cell line was provided by Dr. Julie A. Woods (Ninewells Hospital, University of Dundee, UK). These cell lines were routinely maintained in 75 cm² culture flasks (Sarstedt, Nümbrecht, Germany), in a humid atmosphere of 5% CO₂ at 37 °C [36 (link)]. Cells were grown in 90% RPMI-1640 media containing, 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, Munich, Germany) and supplemented with 30 mg/L penicillin and 40 mg/L streptomycin. Cells were incubated in a 5% CO₂ humidified incubator (Heracell, Thermo Fisher Scientific, Waltham, MA, USA), at 37 °C, and passaged weekly.

The human gastric epithelial cell line AGS (gastric adenocarcinoma ATCC^® CRL-1739^TM) was purchased from the American Type Culture Collection (ATCC, Barcelona, Spain). Cells were grown in Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) (Lonza, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS) of South American origin (Hyclone, GE Healthcare, Logan, UK) and 1% penicillin/streptomycin (5000 U/mL) (Lonza, Madrid, Spain). Cells were plated at densities of ~1 × 10⁶ cells in 75 cm² culture flasks (Sarstedt, Barcelona, Spain) and incubated at 37 °C under 5% CO₂ in a humidified incubator until 90% confluence was reached. The culture medium was changed every two days. Before a confluent monolayer appeared, the sub-culturing cell process was carried out. All experiments were performed between passage 5 and 15 to ensure cell uniformity and reproducibility.

HeLa cells were cultured in 75 cm² culture flasks (Sarstedt, Helsingborg, Sweden) in RPMI 1640 media supplemented with 10% heat-inactivated FBS at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded at a density of 7 × 10⁴ cells/ml and incubated overnight. Prior to treatment, cells were washed once with PBS followed by addition of selenite (5 μM), GS-Se-SG (5 μM) or seleno-DL-cystine (100 μM), dissolved in RPMI 1640 media and incubated for different time-points up to 48 hrs. Culture conditions pertaining to specific experiments have been described in the pertinent sections.