L glutammine

DU-145 and 22Rv1 cell lines were grown in RPMI 1640 medium added of 10% fetal bovine serum, 1% penicillin/streptomycin 2 mM and 1% l-glutammine 2 mM (Euroclone, Milan, Italy). Cells were incubated at 37 °C in a humidified atmosphere containing 6% CO₂. For DCT treatment, cells were grown for 24 h and treated with DCT (Taxotere, 20 mg/mL, Sanofi Aventis, Milan, Italy) for 3 days.

C6 rat glioma cells (ECACC^® 92090409, Sigma Aldrich, Milan, Italy) were cultured in HAM’s F-12 supplemented with 1% L-Glutammine, 10% FBS and 1% penicillin/streptomycin (EuroClone S.p.a., Milan, Italy). Cells were maintained at 37 °C and 5% of CO₂ and sub-cultured as recommended by ECACC^®.

Primary midbrain neurons were obtained from C57BL/6J wild-type mice at embryonic day 12.5 (E12.5) according to previously described protocols [68 (link)]. Briefly, midbrains dissected from E12.5 embryos were washed in HBSS (Euroclone, Pero, Italy) + 1% penicillin/streptomycin (Euroclone). After enzymatic digestion with Accumax (Sigma-Aldrich, St. Louis, MO, USA) and mechanical dissociation in the presence of 0.0625 mg/mL DNAse (Sigma-Aldrich), cells were resuspended in neurobasal medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% penicillin/streptomycin (Euroclone), 1% L-Glutammine (Euroclone), and 2% B27 supplement (Thermo Fisher Scientific). Then, 150 cells/mm² were seeded onto glass coverslips in 24 multi-well plates for immunofluorescence assays or directly on the well for western blot analyses. Both coverslips and wells were previously coated with 0.1 mg/mL poli-D-lysine (Sigma-Aldrich) and 0.01 mg/mL laminin (Sigma-Aldrich). Cells were maintained at 37 °C under a humidified atmosphere of 5% CO₂ in the complete neurobasal medium for 7 days in vitro (DIV), changing half of the medium every two days.