Au l0022

The retinal pigment epithelium (ARPE-19) human cell line was obtained from the American Type Culture Collection (ATCC). ARPE-19 cells and HUVEC isolated from umbilical veins as previously described [21] were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12, Aurogene AU-L0093), glucose 5 mM (Life Technologies A4940-01), supplemented with Hepes 5 mM (Thermo Fisher 15630080), 7.5% NaHCO3 (Thermo Fischer 25080094), 10% inactivated foetal bovine serum (Thermo Fisher 10270106) and 1% penicillin/streptomycin (Aurogene Au-l0022) and maintained at 37°C and 5% CO2. Cells were used from passages 18 to 20 and cultured at a seeding density of 1 × 10⁶ cells/cm³ and experiments repeated three times. Two days after seeding, the culture media was exchanged and supplemented with 1% FBS instead of 10%. Cells were then split as follows, either incubated for 9 d with a high-glucose concentration at 35 mM (HG) or incubated for 24 hours with H₂O₂ (100 µM) as a positive control. Following this stimulation period, cells were treated for 24 hours with the MCR₅ agonist and MCR_3/4 antagonist PG-901 (10⁻¹⁰M) [32,33], MCR₁ agonist BMS (BMS-470539, 10⁻⁵ M) [4], or with the mixed MCR_3/4 agonist MTII (0.30 nmol) [21], at concentrations previously reported in retinal cell cultures [4]. Subsequently, the cells and supernatants were collected and preserved for down stream analysis.

A mean of 3 ml of µFAT was digested in 7 ml of α-Minimum Essential Medium (αMEM; M4526, Merck; Milan, Italy), containing low glucose (5 mM), 1% of l-Glutamine (l-Glu; 25030081, Thermo Fisher Scientific; Milan, Italy), 1% of Penicillin-Streptomycin (P/S; AU-L0022, Aurogene; Rome, Italy) solution and collagenase type II (1 mg/ml, C2-BIOC, Merck; Milan, Italy). After an incubation at 37°C with agitation (200 rpm) for 30 min, the µFAT was filtered through cell strainers (70 μm mesh) and suspended in complete αMEM (5 mM glucose, 1% l-Glu, 1% P/S and 10% Fetal Bovine Serum – FBS; AU-S181H, Aurogene; Rome, Italy) before being centrifuged at 1500 rpm for 5 min at room temperature.³³
The final ASCs pellet was washed three times in Phosphate Buffer Saline (PBS; 14200, Thermo Fisher Scientific; Milan, Italy) before the cell counting. ASCs were grown in complete αMEM at 37°C, 5% CO₂.^{38 (link)}
Particularly, 5 × 10³ ASCs were seeded in 96-well plates to measure cell viability at different time points, while 16.6 × 10³ ASCs/cm² were seeded on culture plates and daily observed with optical microscope until they reached an 80% confluence. Then, ASCs were trypsinized, counted and plated for ASCs characterization by immunofluorescence.