Cytokine/chemokine levels

Hemibrain tissue (n = 4 per group) was snap-frozen after PBS perfusion and stored at −80 °C until analysis. Tissue homogenates from hemibrains were prepared in RIPA-lysis buffer containing 65 mM Tris-base, 150 mM NaCl, 1% Triton-X, 0.25% sodium deoxycholate, 1 mM EDTA, and a mix of phosphatase and protease inhibitors (“phosSTOP” and “Complete, mini, EDTA-free,” Roche Applied Science). BCA Protein Assay Kit (Pierce #23225, ThermoScientific, Waltham, MA USA) was used for determining the total protein concentration. To measure the concentration of cytokines we used the ProcartaPlex® Multiplex Immunoassay (eBioscience, Waltham, MA USA) that uses the Luminex technology (multi-analyte profiling beads) to enable the simultaneous detection and quantitation of multiple cytokines and chemokines per sample, including IFN gamma; IL-12p70; IL-13; IL-1 beta; IL-2; IL-4; IL-5; IL-6; TNF alpha; GM-CSF; IL-18; IL-10; IL-17A; IL-22; IL-23; IL-27; IL-9; GRO alpha; IP-10; MCP-1; MCP-3; MIP-1 alpha; MIP-1 beta; MIP-2; RANTES; Eotaxin; IFN alpha; IL-15/IL-15R; IL-28; IL-31; IL-1 alpha; IL-3; G-CSF; LIF; ENA-78/CXCL5; and M-CSF. All samples were measured in duplicate and the mean values of the two reads were calculated and used in subsequent statistical analysis. Data are presented as pg cytokine/chemokine per mg total protein for the brain lysates.