Non essential amino acids (neaa)

1,2-Dioleoylphosphatidylcholine (DOPC), 1,2-dioleoylphosphatidylethanolamine (DOPE), and DOTAP chloride (USP grade) were obtained from Lipoid GmbH (Heidelberg, Germany). Sorbitan monostearate (SPAN) was obtained from Sigma-Aldrich (Merk, Buchs, Switzerland), and polysorbate 80 (PS80) and squalene (Sq) were obtained from Acros Organics, and coconut oil triacylglycerols (COTs) were obtained from commercial coconut oil.
DMEM, L-glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin, amphotericin B, MTT, trypsin solution in EDTA, Earle’s solution, glucose, and Versene’s solution were purchased from PanEco, Moscow, Russia. DMSO and Triton X-100 were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Fetal bovine serum was purchased from PanEco, Moscow, Russia.

Neuronal cells (20,000) were plated in each well of Matrigel-treated 24-well plates and were cultured in DMEM/F12 medium supplemented with 2% serum replacement (“Gibco”, Carlsbad, CA, USA), 1 mM non-essential amino acids (“Paneco”, Moscow, Russia), 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA), penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia), 1% B27 supplement (“Life Technologies”, Carlsbad, CA, USA), 5 µM forskolin (“Stemgent”, Cambridge, Massachusetts, USA), 20 ng/mL BDNF, 20 ng/mL GDNF and 200 µM ascorbic acid (all from “Peprotech”, Cranbury, NJ, USA) in a CO₂ incubator at 5% CO₂ and 37 °C. On the day of the experiment, the growth medium was changed to DMEM/F12 medium supplemented with 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA) and penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia). N-ADA or N-DDA (5 µM) were added to the cells at the same time point and the control cultures were treated with an equal volume of DMSO. Cells were then incubated for 40 min and treated with 200 µM H₂O₂; the same volume of growth medium was used for the control wells. At 3 and 24 h time points of incubation, the cells were collected for RNA extraction and preparation of cell lysates and the conditioned growth media were collected for ELISA.

Neuronal cells (20,000) were plated in each well of Matrigel-treated 24-well plates and were cultured in DMEM/F12 medium containing 2% serum replacement (“Gibco”, Carlsbad, CA, USA), 1 mM non-essential amino acids (“Paneco”, Moscow, Russia), 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA), penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia), 1% B27 supplement (“Life Technologies”, Carlsbad, CA, USA), 5 µM forskolin (“Stemgent”, Cambridge, MA, USA), 20 ng/mL BDNF, 20 ng/mL GDNF and 200 µM ascorbic acid (all from “Peprotech”, Cranbury, NJ, USA) in a CO₂ incubator at 5% CO₂ and 37 °C. At day 15, the cultures were fixed with 4% neutral-buffered formaldehyde. Immunofluorescence staining was performed according to a previously described method [25 (link)] using β-III-tubulin and tyrosine hydroxylase (TH) antibodies as pan-neuronal and DA neuron-specific markers, respectively. Cells were investigated with an AxioImager Z1 fluorescence microscope equipped with an AxioCam HRM camera using AxioVision 4.8 software (Zeiss, Oberhohen, Germany). The whole surface of each well was imaged for cell counting and the obtained images were analyzed with ImageJ 1.49 software (“NCBI”, Bethesda, MD, USA) using an ITCN plugin (Center for Bio-image Informatics, Santa Barbara, CA, USA).