Poly l lysine (pll)

Six-well plates (Costar Flat Bottom, Corning Inc., Corning, NY, USA) were coated with 0.1% (w/v) poly-L-lysine (Sigma-Aldrich) 1.5 × 10⁶ cells were seeded in a 6-well plate (Corning Costar Flat Bottom) coated with poly-L-lysine. After 5 min, solution was aspirated and the surface was thoroughly rinsed with sterile tissue culture grade water. The plates were allowed to dry at least 2h before introducing cells and medium. Cells (50,000 cells/well) were seeded for overnight and the plate was taken to a 5% CO₂ incubator with IncuCyte S3 imager installed (Sartorius, Essen BioScience, Ann Arbor, MI, USA). Changes in the cellular morphology was continuously visualized using the IncuCyte^® Live Cell Imaging System. This is an automated detection of percent confluency every 2h which enables real-time, live cell counting within a standard cell culture incubator.

Transwell co-cultures were performed as previously described [62 (link)]. BV-2 cells were plated onto the top side of the transwell inserts (0.4 μm pore size polyester membrane precoated with poly-L-lysine; Corning, NY, USA) at the cell density of 3 × 10⁵ cells/ml. The transwell cultures were positioned approximately 2 mm above the HUVECs-enriched cultures and the BV-2 cells grown on the transwells were separated from the HUVECs-enriched cultures by the permeable transwell membrane. Melatonin was diluted to a stock solution of 0.1 mM in 100% DMSO. Then, 1 μg/ml LPS, 100 μM melatonin, LPS plus melatonin or DMSO (100μM) was added to the media below the transwells.

BV‐2 cells were plated onto the upper chamber of transwell inserts (0.4 μm pore size polyester membrane pre‐coated with poly‐L‐lysine; Corning, NY, USA) at a density of 3 × 10⁵ cells/ml. The transwells were positioned approximately 2 mm above PC12 cells. LPS, NBP (30 μM), LPS plus NBP or DMSO as a solvent control were added to the media of PC12 cells for 24 hrs. At the end of each experiment, mRNA and protein were harvested.

Fluorescent-gelatin degradation assays were performed according to the protocol described by Mazurkiewicz et al. [27 (link)]. Coverslips coated with poly-L-lysine (Corning, New York, USA) were washed with PBS and then fixed with 0.5% glutaraldehyde for 15 min. Next, the coverslips were placed on a 30-μl drop of gelatin labeled with fluorescein (Invitrogen, Waltham, Massachusetts, USA), incubated for 10 min, and rinsed with PBS. Then, 5 mg/ml sodium borohydride (Merck, Darmstadt, Germany) was used for quenching the residual reactive groups.
After washing with PBS, cells were seeded in 24-well plates containing the prepared coverslips and incubated at 37 °C in air with 5% CO₂ and 95% humidity. After 16 h, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with Alexa Fluor 568 phalloidin (Thermo Fisher, Waltham, Massachusetts, USA) to visualize filamentous actin. Confocal images were captured using a Leica Stellaris 8 (Leica, Wetzlar, Germany) and LAS X software (ver. 3.3.0, Leica, Wetzlar, Germany). Locations of degraded gelatin were visible as dark areas that lack fluorescence in the bright-green fluorescent gelatin matrix. Experiments were conducted with three repetitions.