Measurement of reactive oxygen species in the culture media using Acridan Lumigen PS-3 assay.
Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at -20°C, in as little as 10-20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.
['Uy|Benedict|B|', 'McGlashan|Susan R|SR|', 'Shaikh|Shamim B|SB|']
D000166:Acridines D000818:Animals D002021:Buffers D002460:Cell Line D002623:Chemistry Techniques, Analytical D017077:Culture Media, Conditioned D004305:Dose-Response Relationship, Drug D006801:Humans D007202:Indicators and Reagents D049449:Luminescence D009000:Monocytes D061251:Primary Cell Culture D051381:Rats D017208:Rats, Wistar D017382:Reactive Oxygen Species D012402:Rotenone D012680:Sensitivity and Specificity
ROS chemiluminescent assay free radicals