Fccp oligomycin

Haloperidol, risperidone, and aripiprazole were acquired from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA) and dissolved in DMSO per manufacturer’s instructions. iNOS (Thermo Fisher Scientific, Waltham, MA, USA) and CD206 (BioRad, Hercules, CA, USA) monoclonal antibodies were used for immunofluorescence. Secondary antibodies used were Alexa 488 goat α-mouse IgG₁, Alexa 488 and 555 goat α-rat IgG_2a and Alexa 555 goat α-rabbit secondary reagents (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies used in flow cytometry analysis were anti-CD86-PE (BD Biosciences, San Jose, CA, USA, SAD) and anti-CD16/32-FITC (BD Biosciences, San Jose, CA, USA, SAD). mTOR signaling was assessed with anti-phospho-p70S6K and anti-p70S6K antibodies (Cell Signaling Technologies, Danvers, MA, USA), while anti β-Actin (Sigma Aldrich, St. Louis, MO, USA) was used as a loading control. Secondary antibody for Western blot analysis was HRP conjugated goat α-rabbit (Cell Signaling Technologies, Danvers, MA, USA). Real-time metabolism assay was performed using IFN-γ (Genentech, San Francisco, CA, USA), LPS (Sigma Aldrich, St. Louis, MO, USA) and FCCP/Oligomycin as part of the Cell Energy Phenotype Test Kit (Agilent, Santa Clara, CA, USA).

Naltrexone hydrochloride (Sigma Aldrich, St. Louis, MO, USA) (−)- naltrexone stereoisomer was used for all experiments and dissolved in purified H₂O as per the manufacturer’s instructions. Tested concentrations for Naltrexone hydrochloride were 50 μM, 100 μM, 250 μM, 500 μM and 1000 μM. For immunofluorescence, we used iNOS (Thermo Fisher Scientific, Waltham, MA, USA) and CD206 (BioRad, Hercules, CA, USA) monoclonal antibodies. Secondary antibodies used were Alexa 488 goat anti-mouse IgG1, Alexa 488 and 555 goat anti-rat IgG2a and Alexa 555 goat anti-rabbit secondary reagents (all from Thermo Fisher Scientific, Waltham, MA, USA). The blue-fluorescent DNA stain DAPI was used for nuclear staining (Thermo Fisher Scientific, Waltham, MA, USA). mTOR signalling was assessed using anti-phospho-p70S6K and anti-p70S6K antibodies (Cell Signalling Technologies, MA, USA), while anti β-Actin (Sigma Aldrich, St. Louis, MO, USA) was used for protein loading control. The secondary antibody for Western blot analysis was HRP conjugated goat anti-rabbit (Cell Signalling Technologies, MA, USA). Real-time metabolism assay was performed using IFN-γ (Genentech, San Francisco, CA, USA), LPS (Sigma Aldrich, St. Louis, MO, USA) and FCCP/Oligomycin as part of the Cell Energy Phenotype Test Kit (Agilent, Santa Clara, CA, USA).