Foetal bovine serum (fbs)

Bone marrow-derived macrophages (BMDMs) were obtained as previously described [28 (link)]. Briefly, cells were obtained from the tibia and femur of adult C57Bl/6 mice and were incubated in Roswell Park Memorial Institute medium (RPMI) 1640 (Gibco, Cat.No. 11875085, Pasley, Scotland) supplemented with 20% (v/v) of foetal bovine serum (Dutscher, Cat.No. S1018-500, Bernolsheim, France) and 30% (v/v) of L929 conditioned medium enriched in macrophage colony stimulating factor, for 7 days at 37 °C in humidified 5% CO₂. Differentiated BMDM were detached, counted, seeded in a tissue culture plate (1.0 × 10⁶ cells/mL) overnight and finally stimulated the following day as described below.

The human glioblastoma cell line U87MG was obtained from ATCC (American Tissue Culture Collection, Rockville, MD, USA); T98G cells were from ECACC (European Collection of Authenticated Cell Cultures, Sigma-Aldritch, Hamburg, Germany). LN443 cells were kindly provided by Prof. Monika Hegi (Lausanne, Switzerland). According to the canSar database, these cell lines express wild-type EGFR [23 (link)]. GBM cells were maintained in Eagle’s minimum essential medium (EMEM) (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum (FBS) (Dominique Dutscher, Brumath, France), 1% sodium pyruvate (Lonza, Verviers, Belgium) and 1% nonessential amino acid (Lonza, Verviers, Belgium), in a 37 °C humidified incubator with 5% CO₂.

Human synoviocytes were recovered from the synovial membrane of six patients undergoing hip replacement surgery (mean age = 75 years). The cells were released by enzymatic digestion of the synovial membrane with collagenase type I (2 mg/ml, 12 h; ThermoFisher, Waltham, USA). The cells were cultured in Dulbecco’s Modified Eagle Medium high glucose with glutamine and sodium pyruvate (DMEM, Dutscher), supplemented with 10% Foetal Bovine Serum (FBS, Dutscher) and penicillin-streptomycin (Lonza), then incubated at 37 °C in a humid atmosphere containing 5% CO₂.
To achieve the desired number of cells, passages were performed. The cells were rinsed with DPBS, then detached with 0.05% trypsin (ThermoFisher). The cells were recovered in a culture medium and seeded at approximately 7500 cells/cm². The absence of mycoplasmas was checked by PCR.
The cells were processed at the confluence stage. Treatments were diluted in a new culture medium to the desired concentration. Each molecule was tested alone or in the presence of LPS. The three molecules were also tested together in order to see the effects of the combination of these three extracts, in the presence and absence of LPS.