Bioplex 100 instrument

Brain region samples (NAC, PFC, VTA, and AMG were stored at −80° C in 1.5 ml microcentrifuge tubes, and were extracted for analysis by mechanical homogenization in 150 µl of 4° C PBS with Kontes^® microcentrifuge pestles, followed by mixing with an equal volume of Cell Lysis Buffer 2 (R&D Systems, Minneapolis, MN) and incubation for 30 min at room temperature with gentle agitation. Samples were then centrifuged at 12,000 x g at 4° C for 10 minutes, and supernatants were transferred to new microcentrifuge tubes. Immediately after centrifugation, total protein content (mg total protein/ml) was determined using the Pierce BCA Protein assay (ThermoFisher, Waltham, MA). Samples were stored at −80° C until assayed further. Cytokine/chemokine levels for rat IL-1β, IL-6, IL-10, IL-17A, CCL2/MCP-1, CCL5/RANTES, and TNF-α were determined by Milliplex^® Luminex^® multiplex assays (Millipore/Sigma, Burlington, MA). Luminex^® assays were conducted using a BioRad BioPlex100 instrument. Levels of rat CXCL12/SDF-1α were determined by ELISA (Novus Biologicals, Centennial, CO). The ELISA assays were read using a FluoSTAR Omega spectrophotometer (BMC Labtech, Cary NC). Pierce BCA Protein assays, Milliplex^® assays, and ELISA assays were conducted following the protocols included with each kit.