Multiplex Cytokine Profiling in Brain Cortex

The protein levels of a panel of inflammatory cytokines were measured in the neocortex by Meso Scale Discovery (MSD) multiplex immunoassay (sector imager 2400, Meso Scale Discovery; Gaithersburg, MD) as previously described [38 (link)]. Brain cortex was homogenized using high shear homogenizer (Omni TH115), in a 1:10 (w/v) of ice-cold lysis buffer consisting of PBS containing 1 μg/ml Leupeptin, 1 mM PMSF, and 1 mM EDTA. The cortical homogenate was centrifuged at 14,000 × g for 20 min at 4 °C in a microcentrifuge. Fifty microliters of the resulting supernatant was loaded per well of the custom MSD plate, and cortical cytokine levels were determined by MSD assay (Mouse Proinflammatory 7-Plex Ultra-Sensitive (K15012C)). Cytokine levels in the cortex were normalized to the total amount of protein in the sample loaded as determined by BCA Protein Assay (Pierce).