Quantification of Cytokine Levels

The collected blood was allowed to clot and then it was centrifuged at 1000× g for 10 min. Then, serum samples were collected into clean tubes and stored at −20 °C until used in ELISA assays. The isolated brain prefrontal cortexes were homogenized and lysed using a lysis buffer (Cloud-Clone Corp., Houston, TX, USA), following the manufacturer’s directions. Lysates were centrifuged at 10,000 rpm and 4 °C for 5 min and then they were stored at −80 °C until used. Protein content was determined by Bradford method [110 (link)]. The levels of IL-1β, IL-6, and TNFα, were quantified in a single serum or brain tissue sample using an appropriate for mice ELISA Kit (Cloud-Clone Corp., Houston, TX, USA), as previously described [59 (link)]. We performed the determinations in accordance with the manufacturer’s instruction. The optical density of individual wells was measured with a spectrophotometric microplate reader (BioTek, Elx808, Warsaw, Poland) at a wavelength of 450 nm. The concentration of cytokine in the samples was determined by comparing the optical density of the samples with the standard curve. Cytokine concentrations in the prefrontal cortex are expressed in picograms per ml/mg of protein, and picograms per ml of serum.