Milliplex analysis

For protein analysis, cell culture supernatants were collected on ice from mouse cerebrocortical cell cultures at the indicated time points (3 to 24 h) post-treatment with mIFNβ (500–5,000 U/mL) or BSA/PBS as vehicle control (indicated as 0 h exposure to IFNβ). Samples were centrifuged to remove cell debris and analyzed for 6 different proteins (CCL3, CCL4, CCL5, CXCL10, IFNβ and IFNγ) using a commercially available Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel from Millipore (Billerica, MA, cat # MCYTOMAG-70K) following the supplier’s instructions. Bead-bound protein concentrations were measured with MAGPIX system from Millipore and analyzed by Milliplex Analysis (Millipore, Billerica, MA).

Serum concentrations were measured using commercial kits for free fatty acids (FFA, NEFA HR(2) kit, Wako Chemicals GmbH, Neuss, Germany) and triacylglycerides (TG, Triacylglycerides liquicolor kit, Human Diagnostics, Wiesbaden, Germany). All analyses were performed according to the manufacturers’ protocols, but the volumes were scaled down to allow for analysis with a plate reader (BioTek Synergy HT, Bad Friedrichshall, Germany).
Serum concentrations of insulin, leptin, and adiponectin were measured by Milliplex analysis (Millipore Corporation, Billerica, MA, USA). The sera were diluted 5 times (leptin, insulin) or 5000 times (adiponectin). The assays were conducted according to the manufacturer’s protocol and measured using the Bio-plex 200 system with Bio-plex manager software (Biorad Laboratories, Veenendaal, The Netherlands).
For glucose analysis, the sera were diluted 10 times in 0.3 M trichloroacetic acid (Merck, Darmstadt, Germany) and centrifuged for 5 min at 1750× g. The supernatant was mixed with glucose oxidase solution (1:5; GOD-PAP kit, Roche, Woerden, The Netherlands) in a 96-well plate and, after 30 min incubation at room temperature, the extinction at 490 nm was measured using a 96-well plate reader (BioTec Synergy HT, Bad Friedrichshall, Germany).
Serum samples were measured in duplicate and averaged, and concentrations were calculated using standard curves.