Non essentials amino acids

Cell lines MDA-MB-231 from triple-negative breast cancer and HeLa from cervical cancer were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). They were cultured at 37 °C with 5% CO₂ in DMEM middle (Lonza, Basel, Switzerland), supplemented with 10% fetal bovine serum (Dutscher, Brumath, France), 1% Zell Shield (Dutscher) and 1% non-essentials amino-acids (Lonza). Cancer cells were plated in 96-well plates (2 × 10³ cells/wells) in triplicate, incubated at 37 °C with 5% CO₂ for 24 h, and treated with 1,4-Dihydroquinolines for 72 h at different concentrations. Cell viability was determined using CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS, Promega, Madison, WI, USA). 20 µL reagent was added in each well and cells were incubated for 2 h at 37 °C/5% CO₂. Absorbance was measured at 490 nm (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). Calculations of IC50 were performed using GraphPad Prism V7.0 software (GraphPad Software, La Jolla, CA, USA).

Human primary fibroblasts (75,000 cells) were cultured in 1 ml of DMEM (Lonza) supplemented with FCS 10%, 2 mM L-glutamine, 1 mM nonessentials amino acids (Lonza), 5 U/ml penicillin, and 5 U/ml streptomycin (Pen-Strep (Lonza)), in six wells plates. Forty-eight hours later, 10¹–10⁶ tissue single cells suspensions were added on the fibroblasts. Parasite tissue load was assessed by enumeration of the plaques formed under a microscope, and expressed as the number of plaque forming units per 10⁶ tissue single cells added.

Single-cell suspensions from spleen and MLNs were prepared using standard methods. BM and SILP were processed as previously described^{18 ,36 (link)}. Blood was collected from the retro-orbital plexus after anesthesia and resuspended in 20 µl of heparin (Choay) to prevent coagulation. Cells were washed in RPMI 1640 (Lonza) supplemented with fetal calf serum (FCS) 10%, 2 mM L-glutamine, 25 mM Hepes, 1 mM nonessentials amino acids (Lonza), 5 U/ml penicillin, and 5 U/ml streptomycin (Pen-Strep (Lonza)) (complete medium). Cells were then resuspended in 1 ml of ACK (8.29 g/ml NH4Cl, and 1 g/l KHCO3, 37.2 mg/l Na-EDTA (ethylenediaminetetraacetic acid) dissolved in demineralized water) for 1 min at room temperature, except for the blood, which was resuspended in Red blood cell lysis buffer (Beckton Dickinson) following manufacturer’s instructions. Cells were washed in RPMI complete medium again.