Retinal Explant Culture for Iron Exposure and Transferrin Therapy

Adult Wistar rat retinas were isolated from fresh enucleated eyes, divided into four parts, and transferred to 0.2-mm polycarbonate membranes (Millipore) with the GCL facing the membrane (Fig. 2). Mouse retinal explants were isolated as indicated above, with one explant corresponding to one eye. The insert was placed into a six-well culture plate containing Dulbecco’s modified Eagle’s medium with l-glutamine and 5% serum (Thermo Fisher Scientific) corresponding to day 0. To analyze the effects of iron exposure on retinal explants, 30 μl of medium containing FeSO₄ (7H₂O; Sigma-Aldrich) was added to the PR side of the explants (upper chamber). To explore TF’s therapeutic effects, the medium in the upper chamber was removed after 2 days. PBS was added for washing, and 30 μl of medium containing human apoTF (0.5, 5, or 50 mg/ml; Sigma-Aldrich) was placed on the retinas. LDH release and enzyme-linked immunosorbent assay (ELISA) directed against TF were measured in medium collected from the lower chamber as described previously (12 (link), 13 (link)). Histology, iron staining, immunohistochemistry, and Western blotting were performed on explants as described below and in the time workflow legends.