Htb 14

Human glioblastoma (U87-MG, ATCC^® HTB-14™, Manassas, VA, USA) and human microglial clone 3 (HMC3, ATCC^® CRL-3304™, Manassas, VA, USA) cell lines were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and a 1:1 antibiotic mixture of streptomycin (100 g/mL) and penicillin (100 U/mL) (Sigma-Aldrich, Milan, Italy) at 37 °C in 5% CO₂ humidified air.
Aβ_25–35 peptide (NH₂-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-COOH) (A4559, Sigma-Aldrich, Milan, Italy) was initially dissolved in double-distilled water to obtain 1 mM concentration and stored at −20 °C. To form aggregated diffusible oligomers, the solution was incubated at 37 °C for 5 days [31 (link)], then diluted in medium to the indicated concentration, just prior to cell treatments. FD22a was dissolved in DMSO to obtain a 50 mM stock solution which was kept at 4 °C. Before the experiments FD22a stock solution was diluted into the cell culture medium to the desired experimental concentration, and the final DMSO concentration was maintained no higher than 0.1%. Vehicle-treated cells (0.1% DMSO) were used as control (Ctrl).
In all the experiments 24 h after seeding, cells were exposed to pretreatment with FD22a for 24 h and then exposed to Aβ_25–35 at the pertinent concentration (10 µM for U87-MG and 1 µM for HMC3). After 48 h, cells were processed according to the specific experiment protocol.

U87 MG (ATCC, HTB-14™) is a human malignant glioblastoma–astrocytoma cell line that is classified as grade IV. The cells were adherent epithelial cells and were cultured in DMEM (Dulbecco’s modified Eagle medium) + GlutamaxI (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37 °C under humidified atmosphere.