High glucose dmem medium

The cell lines used in this study were maintained in an atmosphere containing 5% CO₂ at 37 °C in the culture medium recommended by ATCC. Human epithelial lung A549 cells (ATCC, CCL-185) and murine macrophages J774A.1 (ATCC, TIB-67) were cultured in DMEM high glucose medium (Dominique Dutscher, Brumath, France) supplemented with 10% of heat-inactivated fetal bovine serum (Dominique Dutscher). Human pharyngeal epithelial FaDu cells (ATCC, HTB-43) were cultured in MEM medium (Dominique Dutscher) supplemented with 10% of heat-inactivated fetal bovine serum.

All human cell lines purchased from ATCC (LGC Standards, Molsheim, France) Human mammary (MCF-7, ATCC^® HTB-22), pancreatic (AsPC-1, ATCC^® CRL-1682) and colon (SW-620, ATCC^® CCL-227^™) adenocarcinoma cell lines were maintained in DMEM high-glucose medium (Dominique Dutscher, 67,172 Brumath, France, Cat No L0102-500), while the human acute monocytic leukemia cell line (THP-1, ATCC^® TIB-202) was maintained in RPMI-1640 medium (ATCC^® 30-2001™, LGC Standards, Molsheim, France), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Life Technologies, Paisley, UK, Cat No 10270-106) and 1% (v/v) penicillin-streptomycin (10,000 units/mL and 10,000 µg/mL, Life Technologies, Paisley, UK, Cat No 15140-122). Cells were kept at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂ during their exponential growing phase and in the course of incubation with investigated compounds. Before confluence, adherent cells were trypsinized and sub-cultured twice a week.
Investigated extracts were initially dissolved in dimethyl sulfoxide (DMSO) in a concentrated stock solution. Further dilutions to the experimental concentrations applied on the cells have been done in RPMI-1640 or DMEM media prior to each experiment, thus the final concentration of DMSO on treated cells was 0.5% (v/v) for the most applied concentrations.

The MW2 methicillin resistant Staphylococcus aureus (MRSA) strain was provided by the Laboratory of Human Bacterial Pathogenesis, National Institute of Health (Bethesda, MD, USA) and was cultivated as described previously (27 (link)). Briefly, aliquots of frozen S. aureus bacteria were cultivated overnight in Brain Heart Infusion (BHI, Bacto TM BHI, Becton Dickinson) broth at 37°C, then 1 mL of overnight culture was diluted (1:50) in high glucose DMEM medium (Dominique Dutscher, Bernolsheim, France). Strains were grown in 50 mL tubes at 37°C under agitation until cultures had reached an optical density of 0.6 at 600 nm, corresponding to approximately 10⁸ colony-forming unit per milliliter (CFU/mL). Bacteria were harvested by centrifugation and resuspended in the interaction medium (DMEM). Bacterial concentrations were estimated spectrophotometrically and the number of live bacterial cells was confirmed by plate counts on BHI agar.