Human Monocyte Isolation and Stimulation

The study protocol involving the use of human blood cells was approved by the Ethics Committee of the University of Naples Federico II. Cells were isolated from buffy coats of healthy donors. Blood was layered onto Histopaque-1077 (Sigma-Aldrich) and mononuclear cells were collected at the interface. Monocytes were further purified with anti-CD14 Microbeads (Miltenyi Biotec). Purity of cell preparations was >95% as assessed by flow cytometry. Cells were cultured in cIMDM-5 [IMDM, 5% FCS, 1× non-essential amino acids, 1× UltraGlutamine, 25 mM HEPES, 5 µg/mL gentamicin (Lonza)] in 96-well flat-bottom plates (10⁵ monocytes/well) in a final volume of 250 µL. For experiments involving flow cytometry, cells were cultured in suspension (1.5 mL tubes) in cIMDM-5 at a concentration not greater than 2 × 10⁶ cells/mL, then spun down and collected for subsequent experiments.
Cells were treated with different combinations of: LPS (Escherichia coli 026:B6) 10 ng/mL (Sigma-Aldrich), IL-3 5 ng/mL (Peprotech), M-CSF 25 ng/mL, GM-CSF 5 ng/mL (Miltenyi Biotec), P3CSK4 10 ng/mL, Poly(I:C) 1 µg/mL, flagellin 10 ng/mL, imiquimod 1 µg/mL, ODN2006 1 µM (Invivogen), BAY11-7082 1 µM, SP600125 2 µM, rapamycin 50 and 250 nM, torin1 10 and 50 nM, AGK2 10 µM, APO866 0.1, 1, and 10 nM, TG101348 125, 250, and 500 nM (Selleckchem), U0126 2 µM, LY294002 10 µM, SB203580 2 µM (Cell Signaling Technology), 10058-F4 40 µM, EX-527 500 nM (Tocris Bioscience), 2-Deoxy-d-Glucose 1 mM, Etomoxir 40 µM, BAY 85-3934 1 µM, CAY-10585 10 µM (Cayman Chemical), nicotinic acid 10 µM (Sigma-Aldrich), Pyridone 6 100 nM (BioVision), Trichostatin A (TSA) 5 nM (Calbiochem).