2.12. Cytokine measurements

The hippocampus and prefrontal cortex were dissected from mice subjected to chronic restraint stress or non-stressed mice (control) 1, 4 and 24 hr after stress or three hr after the last behavioral test, and homogenized in lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium vanadate, 50 mM sodium fluoride, and 100 nM okadaic acid as previously described (Cheng et al., 2016). Blood samples were quickly collected at the time of sacrifice, centrifuged to obtain the plasma, and stored at −80°C until use. The cytokines TNFα, IL-6, IL-17 and IFN-β were measured by enzyme-linked immunosorbent assay (ELISA; eBioscience or Biolegend) using 150 μg protein according to the manufacturer’s instructions. Briefly, the cytokine-specific capture antibody was immobilized onto an ELISA plate to capture the targeted cytokine, which is then detected by a cytokine-specific biotinylated antibody. The cytokine is quantified using a colorimetric reaction based on activity of avidin-horseradish peroxidase bound to the biotinylated detection antibody. Cytokine concentrations were determined with a microplate reader (SpectraMax M3).