*2.6. Cell Viability Assay*

As previously pointed out, for cell viability study, the cells were seed in 40 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well in poly-d-lysine-precoated 96-well culture plates. Dopaminergic cells were maintained for 7–10 days before performing the experiments and the medium was changed every 3 days, if necessary. At a determined time point, the media from the cells were removed and new fresh media were added with the different nanoparticle concentrations, DHAH-NLCs or Mygliol-NLCs, (100, 75, 50, 25, 12.5 and 5 µM) for 24 or 48 h. The concentration (µM) refers to the quantity of DHAH present in the NLC formulation. For dosing Mygliol NPs, an equal amount of NLCs was used as an internal control to observe the difference between using a functional lipid, such as DHAH, versus an inert lipid, such as Mygliol®.

Afterwards, the viability was assessed using the CCK-8 kit. Briefly, 10 µL of the CCK-8 reagent was added to the cells. After 4 h of incubation, the absorbance of the mixture was read at 450 nm, using 650 nm as the reference wavelength (Plate Reader Infinite M200, Tecan, Männedorf, Switzerland). The absorbance was directly proportional to the number of living cells in the culture. Cell viability for each condition is represented in a percentage related to the control positive (C+), where no treatment was added to the cell media.

In the case of microglia, slight modifications were done in the cell viability assay protocol. After obtaining a pure microglial cell culture, as previously pointed out, the microglia cells were seeded in 50 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well density. Some 24 h after cell attachment, the media were removed and the cells were incubated for 24 or 48 h with the different concentrations of NLCs, as previously pointed out. Then, the viability CCK-8 test was performed as earlier described.
