Trypsin edta

Murine osteosarcoma cell line (MosJ) was used for both in vitro and in vivo experiments. Cells were grown in DMEM high glucose (Lonza Group Ltd., Basel, Switzerland) supplemented with 10% FBS (Eurobio Scientific, Les Ulis, France) and 1% penicillin/streptomycin (Lonza). Depending on the number of cells required for in vitro or in vivo experiments, amplification was performed in either 75 or 175 cm² tissue culture flasks (Corning, Wiesbaden, Germany). Cells were seeded at a density of 1 × 10⁴ cells/cm² and incubated at 37 °C in a humidified tissue culture incubator with 5% CO₂. After 3 or 4 days, when cells reached 70–80% confluence, they were detached using trypsin-EDTA (ethylene diamine tetra-acetic acid) (Eurobio), resuspended in DMEM supplemented with 10% FBS to inactivate enzyme activity, centrifuged at 500 g for 5 min and washed in PBS (Lonza). Cells were counted on a Malassez plate. MosJ cells were either seeded into 24 or 96 well plates for 2D cell culture experiments or injected into para tibial muscles of C57BL/6J mice to give rise to spontaneous osteosarcoma bone tumour growth as previously described [28 (link)].

Caco-2 cells (passages 99 - 106) obtained from the TC7 were cultured in 75-cm² culture flasks (Cellstar cell culture flasks, Sigma-Aldrich) in Dulbecco’s Modified Eagle Medium enriched with glutamine (Gibco, Cergy-Pontoise, France), supplemented with 10% of heat inactivated fetal bovine serum, 0.5% of gentamycine (Eurobio, Courtaboeuf, France) and 1% of non-essential amino acids (Sigma-Aldrich). Cells were maintained at 37 °C in an atmosphere of 5% CO₂ and 90% relative humidity. The medium was changed every 2 days. Cells were passaged once a week. The partially confluent cell monolayers were trypsinized with Trypsin-EDTA (Eurobio).

Melanoma B16F10 and B16OVA cell lines were kindly provided by PR (Ludwig Center for Cancer Research, Department of Oncology—Faculty of Biology and Medicine University of Lausanne, Switzerland). Cells were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal calf serum (10%) (Eurobio), 2 mM  l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M Hepes buffer and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37 °C and 5% CO₂. Cells were grown until 75% confluence and passaged using trypsin/EDTA (ethylene-diamine-tetra-acetic acid) 1 × (Eurobio), washed with PBS and resuspended in supplemented RPMI-1640. Tumor cells were proven Mycoplasma-free using a MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA).