2.4.1. Primary Dopaminergic Cell Culture

Dopaminergic neuronal cultures were prepared from Wistar rat embryos at 15, 16 or 17 days of gestation (E15–E17). Animal procedures were reviewed and approved (3 April 2017) by the Local Ethical Committee for Animal Research of the University of the Basque Country (UPV/EHU, CEEA, ref. M20/2017/019). All of the experiments were performed in accordance with the European Community

Council Directive on "The Protection of Animals Used for Scientific Purposes" (2010/63/EU) and with Spanish Law (RD 53/2013) for the care and use of laboratory animals.

To obtain primary dopaminergic cell cultures, the following protocols were followed with slight modifications [40,41] (Figure 1A). Pregnant rats were euthanized and, under no aseptic conditions, the whole brain was removed from the rat embryos and kept on ice in a Petri dish (10 cm ∅) with HBSS for removing the blood vessels and meninges. Petri dishes containing embryos' brains were put under the magnifying glass to select the brain areas of interest following Gaven et al. protocol [42]. The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for cell preparations. After collecting the brain areas of interest, tissue was incubated with tryspin at 37 ◦C for 15 min under aseptic conditions. Then, it was also incubated with DNase for 30 s and, after that time, the trypsin was inactivated and the tissue was washed twice with DMEM GlutMAXTM FBS 10%, P/S. Then, the tissue was mechanically dissociated by several passages through a 5 and 2 mL pipette. Finally, the cell suspension was passed through a 100 µm nylon strainer and centrifuged for obtaining a cell pellet. The cell pellet was resuspended in neurobasal medium supplemented with 0.5 mM glutamine, 1% antibiotic and 3% B27. Cells were seeded in 96-well culture plates (for the cell viability assay, neuroprotective activity assay and 6-OHDA toxin-induced assay) and in 24-well culture plates with glass coverslips (for the immunofluorescence assay), both previously coated with poly-l-lysine to promote cell adhesion, at a density of 40 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well and 150 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well, respectively. *Pharmaceutics* **2020**, *12*, x 6 of 23 HBSS for removing the blood vessels and meninges. Petri dishes containing embryos' brains were put under the magnifying glass to select the brain areas of interest following Gaven et al. protocol [42]. The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for cell preparations. After collecting the brain areas of interest, tissue was incubated with tryspin at 37 °C for 15 min under aseptic conditions. Then, it was also incubated with DNase for 30 s and, after that time, the trypsin was inactivated and the tissue was washed twice with DMEM GlutMAXTM FBS 10%, P/S. Then, the tissue was mechanically dissociated by several passages through a 5 and 2 mL pipette. Finally, the cell suspension was passed through a 100 µm nylon strainer and centrifuged for obtaining a cell pellet. The cell pellet was resuspended in neurobasal medium supplemented with 0.5 mM glutamine, 1% antibiotic and 3% B27. Cells were seeded in 96-well culture plates (for the cell viability assay, neuroprotective activity assay and 6- OHDA toxin-induced assay) and in 24-well culture plates with glass coverslips (for the immunofluorescence assay), both previously coated with poly-l-lysine to promote cell adhesion, at a density of 40 × 103 cells/well and 150 × 103 cells/well, respectively.

**Figure 1.** Schematic representation of primary cell culture protocols. (**A**) Primary dopaminergic neuron culture isolation and seeding. (**B**) Primary microglia cell culture isolation and seeding. This figure was created using Servier Medical Art templates, which are licensed under a Creative **Figure 1.** Schematic representation of primary cell culture protocols. (**A**) Primary dopaminergic neuron culture isolation and seeding. (**B**) Primary microglia cell culture isolation and seeding. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Commons Attribution 3.0 Unported License (https://smart.servier.com).

2.4.2. Primary Microglia Cell Culture

Spanish Law (RD 53/2013) (8 February 2013) for the care and use of laboratory animals.

Research of the University of the Basque Country (UPV/EHU, CEEA, ref. M20/2017/035). All the experiments were performed in accordance with the European Community Council Directive on "The Protection of Animals Used for Scientific Purposes" (2010/63/EU) (22 September 2010) and with
