3.5.4. 6-Hydroxydopamine-Induced In Vitro Model of Parkinson's Disease

The neuroprotective activities of compounds in 6-hydroxydopamine-induced cell model of Parkinson's disease were examined, as described previously [52].

Neuroblastoma Neuro2a line cells (1 × <sup>10</sup><sup>3</sup> cells/well) were treated with 50 <sup>μ</sup>M of 6-hydroxydopamine (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and, after that, the investigated compounds were added to the neuroblastoma cell suspension at a concentration of 1 and 10 μM. In the other case, the substances were added to the cells 1 h before the addition of the neurotoxin. Cells incubated without 6-OHDA and compounds, and with 6-OHDA only, were used as positive and negative controls, respectively. After 24 h, viability of cells was measured using the MTT method. The results were presented as a percent of positive control data.

#### 3.5.5. Paraquat-Induced In Vitro Model of Parkinson's Disease

Neuroblastoma Neuro2a line cells (1 × 103 cells/well) were treated with compounds at concentrations of 1 and 10 μM for 1 h, and then 500 μM of paraquat (Sigma-Aldrich, St. Louis, MO, USA) was added to the neuroblastoma cell suspension. Cells incubated without paraquat and compounds, and with paraquat only, were used as positive and negative controls, respectively. The viability of cells was measured after 24 h using the MTT method. The results were presented as percent of positive control data.

#### 3.5.6. Reactive Oxygen Species (ROS) Level Analysis in 6-OHDA- and PQ-Treated Cells

Cell suspensions (1 × 103 cells/well) were incubated with compound solutions (10 <sup>μ</sup>M) for 1 h. Then, 6-OHDA at a concentration of 50 μM was added in each well, and cells were incubated for 30 min. In other experiments, cells were incubated with PQ at a concentration of 500 μM for 30 min and 1 h. Cells incubated without 6-OHDA/PQ and compounds, and with 6-OHDA/PQ only, were used as positive and negative controls, respectively. To study ROS formation, 20 μL of 2,7-dichlorodihydrofluorescein diacetate solution (Molecular Probes, Eugene, OR, USA) was added to each well, such that the final concentration was 10 mM, and the microplate was incubated for an additional 10 min at 37 ◦C. The intensity of dichlorofluorescein fluorescence was measured with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany) at λex = 485 nm, and λem = 518 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as a percentage of positive control data.
