Antibiotic antimycotic solution

Colon cancer cell lines used in this study were purchased from American Type Culture Collection (Sigma-Aldrich Corp, St Louis, MO, USA). HT-29 cells were cultured with RPMI-1640 medium (Sigma- Aldrich), and SW620 cells were cultured with L-15 medium (Sigma- Aldrich). All media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Biowest, Nuaille, France). All of the cell lines were grown in 5% CO2 at 37°C in incubators with 100% humidity.
Considering SW620 cell line was more aggressive, and HT-29 was less aggressive than other colon cancer cell lines [3 (link)], both of them were selected in the experiment. Colon cancer cell lines SW620 and HT29 were cultivated in vitro and divided into six groups according to different treated factors, as shown in Table 9.

The colon cancer cell lines used in this study were purchased from American Type Culture Collection. These cell lines included Lovo, SW620, HT-29, HCT-8 and HCT-116. HEK293 cells were obtained from the Cancer Research Institute of Central South University, China. The HT-29 and HCT-8 cells were cultured in RPMI-1640 medium (Sigma-Aldrich Corp., St. Louis, MO, USA); the Lovo cells were cultured in F-12K medium (Sigma-Aldrich Corp.); the SW620 cells were cultured in L-15 medium (Sigma-Aldrich Corp.); the HCT-116 cells were cultured in McCoy's 5a medium (Sigma-Aldrich Corp.); and the HEK293 cells were cultured in modified Eagle's minimal essential medium (Sigma-Aldrich Corp.). All media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Biowest, Nuaille, France). All of the cell lines were cultured in 5% CO₂ at 37°C in incubators at 100% humidity.

Murine embryonic fibroblast (MEF) cells were obtained from muscles of the buttocks of a 17-day murine embryo, as described in Jozefczuk et al. (2012) (link) and cultured in DMЕМ-F12 (Biowest, USA) supplemented with the Biowest Antibiotic-Antimycotic solution (2.0 μl/ml). Human colon carcinoma cells COLO 205 (Sigma-Aldrich, USA) were cultured in RPMI 1640 (Sigma-Aldrich, USA) with heat-inactivated fetal bovine serum (10%), 2 mМ ʟ-glutamine, and gentamicin (40 μg/ml) added. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO₂/95% air in 100 ml flasks.

Placental MSCs were cultured at 5% CO₂ and 37 °C in a humidified CO₂ incubator (Thermo Fisher Scientific GmbH, Dreieich, Germany) under sterile conditions in Dulbecco’s modified Eagle’s medium high glucose (DMEM LG; Lonza, Basel, Switzerland) supplemented with 1% (v/v) 100 mM sodium pyruvate solution (Lonza, Basel, Switzerland), 10% (v/v) fetal bovine serum (FBS; Lonza, Basel, Switzerland), 1% (v/v) antibiotic-antimycotic solution (BioWest, Nuaillé, France), and 1% (v/v) ascorbic acid (Sigma-Aldrich, St. Louis, USA) in 10-cm tissue culture plates (CELLSTAR®, Greiner BioOne, Frickenhausen, Germany). Harvesting of the cells was performed with application of 0.25% trypsin solution.

Commercially available human AT-MSCs (hAT-MSCs; C-12977, PromoCell, Heidelberg, Germany) were obtained at passage 2. Cells were cultured in Gibco^TM αMEM culture medium (Thermo Fisher Scientific) supplemented with 20% of fetal bovine serum (Sigma Aldrich) and 1% of antibiotic/antimycotic solution (Biowest). Cells were seeded on 75 cm² culture flasks at a density of 1.2 × 10⁴ cells/cm² and maintained under standard conditions at 37 °C and a 5% CO₂ in the atmosphere.

The cell lines Bel-7402, SK-hep-1, HepG2, Huh7, Li-7, and LO2 were obtained from the American Type Culture Collection. All lines were cultured in modified Eagle’s minimal essential medium (Sigma-Aldrich Corp., China). All media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Biowest, France). All cell lines were cultured in 5% CO₂ at 37 °C in incubators at 100% humidity.

Murine macrophages (RAW 264.7) (ATCC-TIB-71) were acquired from the American Type Culture Collection (ATCC) biobank. RAW 264.7 cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) without phenol red, supplemented with 10% FBS, 1% antibiotic/antimycotic solution (Biowest, Riverside, MO, USA), and 1% sodium pyruvate (humidified atmosphere with 5% CO₂ at 37 °C). Cells were seeded in 96-well microplates (5 × 10⁴ cells/well) for cell viability and nitric oxide (NO) production assessment. For determination of interleukins’ levels, the cells were seeded in 12-well microplates (5 × 10⁵ cells/well).