Cell energy phenotype test kit

TIANamp Genomic DNA Kit (Cat. DP304) was from Tiangen. SYBR Green PCR Master Mix (2×) (Cat.4913914001) was from Roche. Recombinant mouse M-CSF (Cat. 415-ML) and GM-CSF (Cat. 416-ML) were purchased from R&D Systems. Glycolytic Rate Assay Kit (Cat. 103346-100), XF cell Mito Stress Test Kit (Cat. 103010-100) and Cell Energy Phenotype Test Kit (Cat. 103275-100) were from Agilent. Glucose Assay Kit (Cat. AB169559), Citrulline Fluorometric Assay Kit (Cat. AB273309), Ornithine Fluorometric Assay Kit (Cat. AB252903), and Adipogenesis Colorimetric/Fluorometric Assay Kit (Cat. AB102513) were form Abcam. Nitric Oxide Assay Kit (Cat. S0021S), Nitric Oxide Synthase Assay Kit (Cat. S0025) and Mito-Tracker Green (Cat. C1048) was from Beyotime. Bis Benzimide Hoechst NO33342 (Cat. B8040) was from Solarbio. Arginase Activity Assay Kit (Cat. MAK112) was from Sigma-Aldrich.

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).

ECAR and OCR were measured using the Seahorse XFe96 analyzer (Agilent) with Cell Energy Phenotype Test Kit. The day before, 10,000 HUVECs/well from each treatment were seeded in a gelatin-precoated 96-well plate and cultured in EBM with the corresponding glucose concentration at 37°C, 5% CO₂, and 8% O₂. On assay day, cells were washed with 200 μl assay medium (Seahorse XF Base Medium with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose) and incubated in 180 μl of fresh assay medium for 1 h at 37°C before the assay. The Cell Energy Phenotype Test consists of three baseline measurements of OCR and ECAR followed by five stressed measurements. The latter are induced by the simultaneous addition of oligomycin, a complex V inhibitor, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupling agent. Oligomycin and FCCP were added to each well to a final concentration of 1 mM and 0.5 mM, respectively. Results were analyzed with Agilent’s Wave software and normalized to cell number. The cell number of each well was determined by adding Hoechst to a final concentration of 2 μM. Then, nuclei were counted using BioTek Cytation 1 Cell Imaging Multimode Reader. Nine biological replicates were used per condition, each one with multiple technical replicates.