Rtebm basal medium

For in vitro studies, human RPE cells (LONZA) were used in the fourth passage, seeded at a density of 50000 cells/cm² on Transwell^® polyester membrane inserts with 6.5 mm diameter and 0.4 μm pore size (Corning^®) and maintained in RtEBM^® Basal Medium (LONZA), supplemented with L-Glutamine, FGF-B, and Gentamicin and Amphotericin-B. During the first 3 days, 2.5% fetal bovine serum (FBS) was also included. The medium was replaced every 3–4 days. To study the Scribble protein expression during RPE cell development and maturation, samples at three different time points of culture were collected: 7, 14, and 21 days in culture (DIC). Subsequently, to study the expression of this protein in pathological conditions, once cells reached 21 DIC, they were incubated with the different types of blood serum obtained from patients with ophthalmological problems.

ARPE-19 cells (CRL-2302) were purchased from ATCC (Manassas, VA, USA) and primary human fetal retinal pigment epithelial cells, H-RPE (00194987) were from Lonza (Basel, Switzerland). DMEM:F12 (11320–033) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). RtEGM BulletKit (00195409) which is a culture system containing RtEBM Basal medium (00195406) and RtEGM Single Quots Supplements (00195407) was purchased from Lonza. ARPE-19 cells were cultured in DMEM:F12 medium containing 100 U/ml penicillin and 100ug/ml streptomycin supplemented with 10% FBS. H-RPE cells were cultured in RtEBM containing Single Quots supplements with the addition of 2% FBS for the first 24hrs or serum free thereafter. Cells were cultured as described until they reach confluent state. Upon confluence, serum was depleted for 24 hours and cells were treated appropriately for another 24 hours. Acadesine was added 1hour before TNF-α and 5-IODO or DPY were added 1 hour prior to Acadesine. Experiments were performed on ARPE-19 at passage 6–9 and on H-RPE at passage 3–4.