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Figure 1 | BMC Pharmacology

Figure 1

From: 15-deoxy-delta 12, 14-Prostaglandin J2 prevents reactive oxygen species generation and mitochondrial membrane depolarization induced by oxidative stress

Figure 1

Degradation of H 2 O 2 and its effect on cellular viability. (A): Degradation of H2O2 in cell cultures. ARPE-19 cells were plated in a 24-well plate (140,000 cells/well) for a day, fed with serum-free medium for a day, then treated with 700 μl of H2O2 (2 mM, 5 mM or 10 mM) for various periods of time, then the concentration of H2O2 remaining in each culture was determined. The OD at 240 nm is a measure of H2O2 concentration (See Methods). Results indicated that H2O2 decreased in each culture in a time-dependent manner, which could be observed clearly in cultures treated with 5 mM or 10 mM H2O2. Readings from cells treated with 2 mM H2O2 were very close to background levels. (B): Time-dependent degradation of H2O2. Results of cultures treated with 5 mM or 10 mM H2O2 were re-plotted and expressed as a percentage of its original concentration. The half-life for H2O2 degradation was ~1 hour under these experimental conditions. (C): Degradation of H2O2 as a function of volume. Cells grown in 24-well plates were treated with 10 mM H2O2 (700 μl/well, 1,400 μl/well or 2,100 μl/well), and then the H2O2 concentration in each well was determined. Results indicated that H2O2 degradation rate was affected by the volume used in each well, such that the concentration remained higher in cultures with higher volume of H2O2 solution. The H2O2 concentration remained constant in culture wells without cells as indicated by the upper-most line (solid diamonds), which was recorded at time zero. Almost identical readings were recorded 1 hour or 2 hours later (not shown). Note: Treatment of cells with H2O2 for 0 hour was determined by applying H2O2 solution to the cultures briefly (~2 min). The solution was then removed, and the OD measured. This short contact with cultures caused a ~10% loss of H2O2 from its original concentration. This can be seen from the difference between "no cells, 0 hr (solid diamonds, the upper-most line) and "0 hr" (open circles, the second line from the top). (D): Cytotoxicity of H2O2 on ARPE-19: Effects of concentration and volume. Cells plated in a 96-well plate for two days were treated with 1 mM, 1.5 mM or 2 mM H2O2 in various volume for 1 day, then the viability of each treatment was determined by the MTT assay. Results indicate that at a fixed concentration, the volume used in each well affected the cytotoxicity of H2O2.

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