Dulbecco s modified eagle medium dmem f12

The lipids 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (PE-Rhod) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The solvents chloroform and ethanol were purchased from Merck Millipore (Burlington, MA, USA). Graphene oxide (GO) was purchased from Graphenea Inc., Cambridge, MA, USA; as reported by the producer, sulfur and nitrogen contents were, respectively, below 3% and 1% in composition. The phosphate buffer saline (PBS) tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). All experiments were carried out with ultrapure water (18.2 mΩ·cm at 25 °C, total organic carbon (TOC) lower than 5 parts per billion (ppb), Ultrapure Millipore^® Water Type, Burlington, MA, USA). Dulbecco’s modified eagle medium (DMEM)-F12, DMEM high glucose, penicillin-streptomycin solution, L-glutamine, fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), and retinoic acid (RA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Calf jejunal tissue was harvested from a slaughterhouse in sterile conditions and immersed in phosphate-buffered saline (PBS) solution supplemented with 1% antibiotic-antimycotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The sample was washed in ice-cold PBS (Sigma-Aldrich, St. Louis, MO, USA), and the mucosa was minced (2 cm²) using a surgical scalpel, then treated with collagenase I (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 mg/mL Dispase (Sigma-Aldrich, St. Louis, MO, USA) for 45 min. The resulting suspensions were filtered (74 µm nylon mesh) and centrifuged at 200× g at 4 °C for 7 min. The cell pellet was re-suspended in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% antibiotic-antimycotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% non-essential amino acids (NEA, Sigma-Aldrich, St. Louis, MO, USA), 15 ng/mL EGF (epidermal growth factor; Sigma-Aldrich, St. Louis, MO, USA), and 1 µg/mL hydrocortisone (Lonza, Basel, Switzerland), and incubated at 37 °C with 5% CO₂. After 72 h, the medium was changed, and 7 days later, the first passages were performed.

All the solvents, vinyl acetate, Azobisisobutyronitrile (AIBN), sodium hydroxide, N,N′-Dicyclohexylcarbodiimide (DCC), N,N-Dimethylpyridin-4-amine (DMAP), Rhodamine B, Folic acid, All-trans Retinoic acid (ATRA), were purchased from Sigma-Aldrich (Merck Group, Milan, Italy).
For the cellular experiments, Dulbecco’s modified eagle medium (DMEM)-F12, DMEM high glucose, penicillin-streptomycin solution, l-glutamine, foetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck Group, Milan, Italy). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent) was obtained from AppliChem ITW Reagents (Nova Chimica Srl, Milan, Italy).

The peptide QDNSRYTHFLTQHYDAK-NH₂ (Ang1-17) and the acetylated analogous peptide Ac-QDNSRYTHFLTQHYDAK-NH₂ (AcAng1-17) were supplied by Caslo Aps, Lyngby, Denmark. All other chemicals, of the highest available grade, were purchased from Sigma-Aldrich (Munich, Germany) and used without further purification. For the cellular experiments, Dulbecco’s modified eagle medium (DMEM)-F12, penicillin-streptomycin solution, L-glutamine, fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS) and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA).