Brain Cytokine Quantification using V-PLEX

Sample preparation for V-PLEX Proinflammatory Panel 1 Mouse kit (Meso Scale Diagnostics, Rockville, MD) was described previously [32 (link)]. Brains were collected and snap-frozen in isopentane then stored at −20 °C until time of homogenization. Brains were homogenized in RIPA buffer (Thermo Fisher Scientific, Waltham, MA) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). Samples were centrifuged at 13,300 RPM for 15 min at 4 °C. Supernatant was collected and stored at −20 °C. Pierce BCA Protein Assay was performed to determine total protein concentration. Samples were diluted and loaded on the MSD plate at the same total protein concentrations. Manufacturer’s protocol for the V-PLEX Proinflammatory Panel 1 Mouse kit was followed as described. Plates were read with Meso Quickplex SQ 120 (Meso Scale Diagnostics, Rockville, MD) and data was analyzed via MSD Discovery Workbench software (Meso Scale Diagnostics, Rockville, MD). Concentrations of cytokines are presented as pg/mL.