Golgiplug with brefeldin a

Microglia isolated from 9-mo-old OPN-KO.5XFAD mice were seeded into 12-well plates at 3×10⁵ cells/well in a conventional microglia culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin/ streptomycin + 10 ng/mL recombinant mouse M-CSF). Microglia were preincubated with 10 μM of a selective αVβ3 inhibitor (Cilengitide, Selleck, Cat. S6387) for 1 h followed by the addition of 12.5 μg/mL rmOPN and cultured for 24 h. BD GolgiPlug with brefeldin A (1 μL/mL, BD Biosciences) was added into culture for the last 4 h before analysis of CD11c⁺ microglial expression of TNF-α by flow cytometry (CytoFLEX LX, Beckman Coulter).

Microglia isolated from 9-mo-old 5XFAD mice were incubated with anti-OPN mAb (clone: MPIIIB10, DSHB) at increasing concentrations (5, 10, 20 μg/mL) for 24 h. BD GolgiPlug with brefeldin A (1 μL/mL, BD Biosciences) was added to cultures for the last 4 h before flow cytometric analysis of TNF-α production by CD11c⁺ microglia. Incubation of microglia with isotype control (mouse IgG1) was included as a negative control.