The amount of hemoglobin released from the human RPE was determined with an enzyme-linked immunosorbent assay (ELISA; Bethyl Laboratories).
22 For this purpose, seven pairs of freshly enucleated (within 24 hours of death) human globes were obtained from the eye bank (age, 57–70 years). Anterior segment, vitreous, and retina were removed exposing the RPE cells. Cyanoacrylate glue was used to seal the suprachoroidal space. Eye cups were then washed gently three times and incubated in a humidified atmosphere of 5% CO
2 and 95% air at 37°C and maintained in Dulbecco’s modified Eagle’s medium (DMEM H16; Invitrogen), 100 IU/mL penicillin G, 100 μg/mL streptomycin, 5 μg/mL gentamicin, and 2.5 μg/mL amphotericin B for 16 hours. At the end of the incubation period, culture media were collected from all experimental groups and centrifuged, and the amount of hemoglobin in the media was determined with an ELISA kit, according to the manufacturer’s instruction.
17 A similar method has been used to determine human extracellular hemoglobin amounts reliably.
23 Briefly, microtiter plates were coated with sheep anti-human panhemoglobin antibody (1:50,000 dilution; Bethyl Laboratories), washed, blocked, and incubated with culture medium and incubated for 1 hour at room temperature. After incubation samples were removed, and plates were incubated with horseradish peroxidase-conjugated sheep anti-human hemoglobin antibody at 1:50,000 dilution for 60 minutes. Detection was accomplished adding the chromogenic substrate 3,3′,5,5′,-tetramethyl-benzidine (Bethyl Laboratories) to the wells. The chromophore development was terminated after 30 minutes with 2 N H
2SO
4. The plates were read at a wavelength of 450 nm with a microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA). Amount of hemoglobin was calculated using the normalization curve obtained from wells containing dilutions of human hemoglobin ranging from 16 ng/mL to 1 mg/mL.