Oxidative stress and short-chain Cer cell viability assays

ARPE19 cells (1 × 10⁴) were plated in 96-well plates and grown at 37°C for 24 h. They were cultured in serum-free medium containing 100–1,000 μM H₂O₂ (Santoku Chemical Industries Co., Tokyo, Japan). After 24 h of incubation, cell viability was assessed by using a CellTiter 96 aqueous nonradioactive cell proliferation assay kit (Promega, Madison, WI). In this kit, viable and living cells are quantified by measuring the absorbance of the assay plates at 490 nm following the addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), which is reduced by metabolically active cells to yield a soluble formazan product. From preliminary and previous studies, we estimated that at least 800 μM of H₂O₂ were needed to induce measurable cell death within 24 h in ARPE19 cells (35). We used an application of 800–1,000 μM of H₂O₂ for 24 h to induce oxidative stress in ARPE19-ASAH1 cells, and ARPE19 cells served as controls. For biochemical and molecular analyses, we treated the cells with 1,000 μM of H₂O₂ for 3 and 24 h, and harvested and froze the cells. Deionized water in place of H₂O₂ served as the vehicle (Veh) for Veh-treated groups. Additionally, ARPE19 cells were grown per the methods stated above and were treated with exogenous C2-Cer dissolved in ethanol (Avanti Polar Lipids, Alabaster, AL) at concentrations of 10–50 μM, and an equivalent amount of ethanol-treated (100%) cells served as controls. Estimations based on preliminary data suggested that 20 μM of C2-Cer was sufficient to induce measurable cell death within 24 h in ARPE19 cells. ARPE19 and ARPE19-ASAH1 cells were then treated with 10–20 μM of C2-Cer for 24 h and cell viability was determined per the methods stated above. To supplement the CellTiter viability assay, cell viability following H₂O₂-induced oxidative stress, as described above, was determined indirectly in a repeated experiment by measuring released lactate dehydrogenase (LDH) into the medium using a commercial kit from Promega (CytoTox-One homogenous membrane integrity assay kit) following the manufacturer’s instructions. For the LDH release assay, fluorescent measure of the release of LDH from cells with a damaged membrane was calculated by subtraction of culture medium background and the cell viability (percent) was calculated from 100% viability (mean value of untreated cells) to 0% viability (mean value of 2% Triton X-100-treated cells).