4.3. Silencing of the Gene Coding for y+LAT2 Transporter

To down-regulate the y⁺LAT2 transporter, transfection with a mix of four types of siRNA duplexes, consisting of 21 nucleotides was performed. Each type of siRNA was targeted to different gene region to obtain the most effective gene silencing. Three-week astrocytes were washed with PBS solution, trypsinized to detach cells and then seeded at a density of 0.7 × 10⁵ per well in 24-well culture plates in 0.4 mL of astrocytic growth medium. On the same day, cells were transfected with 5 nM of siRNA and 3 µL of HiPerFect transfection reagent (Qiagen Benelux BV, Venlo, the Netherlands)/per well, according to fast-forward protocol, designed for adherent cells and provided by manufacturer (Qiagen). The mock control (transfection procedure without addition of siRNA) were used to indicate whether the transfection process results in cytotoxicity or other non-specific effects. Cells were cultivated with the transfection complexes under normal growth conditions for 48 h and then used for further experiments.