Mem 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.

Glioblastoma U87-MG cancer cells were purchased from ATCC (HTB-14), U373 (Uppsala) cells were obtained from ECACC (Sigma Aldrich) and LN443 cells were kindly provided by Pr. M Hegi (Lausanne, Switzerland). All these cells are grown in MEM medium (L0430-500, Dominique Dutscher) with 10 U.mL^-1 penicillin, 100 µg mL⁻¹ streptomycin, 250 U mL⁻¹ fungizone, 1 mM Na-pyruvate, 2 mM glutamine and 10% FBS. Human brain astrocytes were purchased from Alphabioregen (HBMP202) and cultured in their adapted medium (AGPM-03, Alphabioregen). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Cells were cultured and then treated once they reached 70–80% of their confluency inside the plates.

The most active compounds were tested for their cytotoxicity using the Vero cell line (monkey epithelial cell line), according to a previously described method [35 (link)] with some modifications. The cells were cultured in MEM medium (Dutscher, Brumath, France) supplemented with 10% fetal bovine serum (Dutscher), 0.7 mM glutamine and 100 μg/mL gentamicin.
The cells were seeded at 10⁴ cells per well in a 96-well plates and incubated during 24 h. Then, cells were incubated for additional 48 h with the drugs. Cell proliferation was measured with MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, Sigma, Saint-Quentin Fallavier, France) which is added to each well for 1 h at 37° C and 5% CO₂. Subsequently, DMSO was added to the wells containing the cells and MTT to dissolve the formed crystals. The plates were then read to determine the absorbance at wavelength of 540 nm (µQuant, BioTek, Illkirch, France). Cell proliferation was calculated from at least three independent experiments using GraphPad Prism software 7 (GraphPad Software, San Diego, CA, USA). The cytotoxic/antiplasmodial activity ratio corresponds to the selectivity index.