ARPE-19 Cells Respond to High Glucose and DHA

Human retinal pigment epithelium (RPE) cells (ARPE-19) were purchased from the American Type Cell Culture (ATCC–CRL–2302, Manassas, VA, USA) and cultured in Advanced DMEM/F12 basal medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 20% fetal calf serum (FCS, Merck Life Science S.r.l., Milano, Italy) and 100 U/mL penicillin–streptomycin (Gibco™, Thermo Fisher Scientific, Inc., Waltham, MA, USA), with physiological glucose levels (5 mM). During growth, cells were maintained in a humidified environment (5% CO₂). Further sub-cultures of cells (passage number 12) were performed at the proper density in accordance with each experimental method. To investigate the effect of the high glucose concentrations on ARPE-19 metabolism, cultured cells were treated as previously reported [25 (link)]. A 50 mM concentration of D-Glucose was added to cells for 20 h, followed by the administration of docosahexaenoic acid (DHA) for a further 16 h (Cayman Chemical, Ann Arbor, Michigan, MI, USA—50 mg in 200 µL ethanol), and this was then complexed to fatty acid-free bovine serum albumin (FAF-BSA, Merck Life Science S.r.l., Milano, Italy), prior to use as described [28 (link)], to reach the final concentration of 60 µM of DHA.