Sodium puryvate

SW13 cells were cultured in DMEM-1x (Gibco) having 10% foetal bovine serum (FBS), 1% penicillin-streptomycin (Pen-Strep), 1% non-essential amino acids (Gibco), 0.1% Glutamax (Gibco), and 1% Sodium Puryvate (Gibco). SH-SY5Y cells were cultured in RPMI (Euroclone) having 10% FBS and 1% Pen-Strep. SKOV-3 cells were cultured in RPMI medium including 1% Glutamax and 10% FBS. SW13 cells were transiently transfected with Mito-GFP, GFP and H2B-mCherry plasmids by lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Stable cell line generation: 600.000 SH-SY5Y cells were plated on 60 mm plates and transfected with 0.5 µg of H2B-GFP plasmid. After 48 h, cells were transferred into 100 mm plates and cultured in RPMI complete medium including G418/ Geneticin. 15 days later, positive clones were selected.
Transduction of SH-SY5Y cells with a lentiviral vector expressing pCMV-Mito-DsRed: 600.000 SH-SY5Y cells were plated in 60 mm plates. After 24 h, they were infected with 800 µl of LV-CMV-Mito-DsRed. After 48 h, cells expressing Mito-DsRed have been validated.

Cells from a human type II alveolar epithelial cell line (A549, obtained from the American Type Culture Collection, ATCC, Manassas, USA) were cultured in DMEM (Dulbeccos´s Minimal Essential Medium, Cat. No. 41965–039, Gibco^® Invitrogen) cell culture medium supplemented with 10% Fetal Bovine Serum (European grade, Biological Industries), 1 mM Sodium Puryvate (Gibco^® Life Technologies), 100 units mL^-1 Penicillin and 100 μg mL^-1 Streptomycin (Pen Strep, Gibco^® Life Technologies). The supplemented medium is denoted as DMEM⁺. The cells were cultured in cell culture flasks in a humidified (RH > 99%) CO₂-atmosphere (5%) at 37°C. The cells were seeded 24 h prior to each assay at concentrations of 0.08·10⁶, 0.04·10⁶ and 0.02·10⁶ cells cm^-2 for 4, 24 and 48 h exposure times, respectively, in order for the (control) cells to reach confluence in the end of each exposure. CuO nanoparticles (20–40 nm diameter, Sigma-Aldrich), dispersed in DMEM⁺ at concentrations of 20 or 40 μg cm^-2, were used as positive controls in all cellular assays.