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In a set of analogous experiments, ARPE-19 cells treated with various concentrations of H 2 O 2 for 4 hours or 24 hours showed that a 4-hour treatment with 1 mM H 2 O 2 did not affect viability.
Cells were plated in 96-well plates (human primary RPE: 10,000 cells/well, ARPE-19: 15,000-20,000 cells/well, unless otherwise stated) overnight, fed with serum-free medium or treated with testing agents (prepared in serum-free medium) the next day followed by H 2 O 2 treatment for a day, then the viability from each treatment was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [ 27 ] . Culture medium was removed after treatment and then 100 μl MTT solution (100 μg/ml prepared in DMEM) was added to each well.
Though the H 2 O 2 titration curve in ARPE-19 was not as steep as observed in primary RPE, one-day treatment of these cells with 1.8 mM H 2 O 2 could decrease the cellular viability to ~10% of control while 1 mM H 2 O 2 did not affect cellular viability (Fig.
ARPE-19 is a spontaneous occurring, non-transformed RPE cell line which expresses RPE specific markers (such as CRALBP and RPE65) and exhibits morphological polarization and tight junctions similar to native RPE [ 25 26 ] .
Subsequent experiments with immunofluorescent staining indicated that 1.5 mM H 2 O 2 treatment of ARPE-19 cells for 4 hours led to the formation of 4-HNE protein adducts both in the cytoplasm and nucleus, presumably as a result of lipid-peroxidation (Fig.