Hepes

The THP-1 cell line (passages 6–20, ATCC: TIB-202) was cultured in RPMI 1640 medium (Corning, NY, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin (Corning, NY, USA) 10 mM HEPES (Dominique Dutscher, Bernolsheim, France), 2 mM L-glutamine (Corning, NY, USA), and 10% (v/v) heat-inactivated fetal bovine serum, FBS (Corning, NY, USA) at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂. Routinely, THP-1 cells were cultured in T75 flasks and sub-cultured every three to four days at a concentration of 4 × 10⁵ to 1 × 10⁶ cells/mL. THP-1 cells can be differentiated into macrophage-like cells using 100 ng/mL phorbol 12-myristate 13-acetate PMA (Sigma-Aldrich, Milan, Italy) for 72 h. During this time, cell attach to the bottom of the cell culture plates and develop macrophage-like morphology. After macrophage differentiation, cells rest for another 24 h in the culture medium without PMA to obtain the resting state of macrophages (M0).

Intestinal epithelial cells Caco-2, TC7 clone, were cultivated in DMEM medium supplemented with 20% heat-inactivated fetal calf serum (FCS, Dutscher), 2 mM of glutamine (Gibco), vitamins (1%, Dutscher), amino acids (1%, Fisher) and antibiotics cocktail (penicillin, streptomycin, Amphotericin B, 1%, HyClone). Intestinal epithelial cells T84 were cultivated in DMEM/F12 medium supplemented with 10% heat-inactivated fetal calf serum (FCS, Dutscher), 2 mM of glutamine (Gibco), vitamins (1%, Dutscher), 10 mM of Hepes (Dutscher) and antibiotics cocktail (penicillin, streptomycin, Amphotericin B, 1%, HyClone). Cells were seeded into 24-well culture plates at a density of 1.10⁵ cells/well (Caco-2) or 4.10⁵ cells/well (T84) in a medium depleted of antibiotics and were incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 h. Cells were treated with suberoylanilide hydroxamic acid (SAHA, Sigma), a global HDAC inhibitor, prepared in DMSO at different doses (0.4/0.8/1.6/3.125/6.25/12.5 µM) during 24 h.

The human brain endothelial cell line hCMEC/D3, an immortalized suitable in vitro model for BBB, was kindly provided by Dr. Pierre-Olivier Couraud, Institut Cochin, Paris. hCMEC/D3 was cultured following the protocol previously reported [17 (link),18 (link)]. Cells were plated on rat tail collagen I (Life Technologies, Carlsbad, CA, USA) pre-coated Petri dishes (Life Technologies, USA), or Lab-Tek chamber slides (Dutscher, Bernolsheim, France). The cells were seeded at a density of 25,000 cells per cm² and grown until the cells reached 90 to 95% confluence.
Cells were cultured at 37 °C, 5% CO₂ atmosphere in EBM-2 basal medium (Lonza) containing 5% fetal calf serum (FCS), 10-mM HEPES (Dutscher, Bernolsheim, France), 1 ng/mL of basic fibroblast growth factor (bFGF) (Sigma-Aldrich, St. Louis, MO, USA), hydrocortisone (1.4 µM), ascorbic acid (5 µg/mL), chemically defined lipid concentrate (CONC, Life Technologies, USA), and 1% antibiotics (penicillin–streptomycin). The medium was supplemented with 10 mM lithium chloride and replaced every 3 to 4 days. The cells became completely confluent within 4 days. All experiments were performed on cultures of confluent cells between passage 29 and 35. Periodically, the culture was tested for mycoplasma contamination using the MycoProbe^® Mycoplasma Detection Kit (Catalog # CUL001B, R&D Systems, Abingdon, UK).