Ultrastructural Analysis of NPS-P88 Internalization

The hCMEC/D3 cells and the T98G cells were seeded in 6-well plates at a density of 3,5 x 10⁴ cells per well. Once they reached confluence, these cells were stimulated with or without 20 ng/mL of IL-1β (Cat. #579404, Biolegend) for three hours at 37°C in 5% CO₂. After the stimulation period, the NPS conjugated with P88 (1 mM) diluted in the culture medium, were added to the cell wells and incubated for three hours at 37°C in 5% CO₂. Following the incubation time, the cells were washed three times with PBS and were trypsinized and centrifuged at 1200 rpm for 10 minutes. Then, the pellet was diluted in glutaraldehyde 2.5%. Once the cells were fixed, they were centrifuged at 13,000 rpm for 3 minutes. Subsequently, a post-fixation process was carried out using 1% osmium tetroxide in water for two hours at 4°C, followed by a pre-imbibition step with 3% uranyl acetate for 1 hour at room temperature. The dehydration process involved a series of ethanol gradients at different concentrations: 50%, 70%, 90%, 100%, 100%, each lasting 10 minutes. This was followed by acetone-ethanol (1:1) for 15 minutes and then acetone for another 15 minutes. For the SPURRs epoxy resin, the following procedure was followed: Mix Resin Spurr with acetone (2:1) for 1 hour, Mix Resin Spurr with acetone (1:1) for 1 hour, Pure Resin Spurr for 2 hours, and polymerize for 12 hours at 72°C. The samples were sectioned using a Leica EM UC7 ultramicrotome, creating slices with a thickness of 130 nm. These slices were contrasted with 6% uranyl acetate and lead citrate. Finally, the samples were observed using a JEOL 1400 plus TEM, and images were captured using a Gatan Orius CCD camera.