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Induction and Inhibition Studies of Hypoxia and Oxidative Stress in Immortalized KeratinocytesDownload
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January 21, 2014
Authors: Jens Roesler, Brad Larson, and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont; Stephanie Georgiou, Enzo Life Sciences, Farmingdale, New York
Hypoxia is a pathological condition where the entire body, or a portion of the body, is deprived of adequate oxygen supply. Variations in oxygen concentration can be part of normal physiology; such as during strenuous exercise, high altitude climbs, or deep sea dives. However, hypoxia can also be a serious condition. Many children born prematurely experience generalized hypoxia due to the fact that their lungs have not fully developed, and oxygenated blood is not adequately distributed throughout the body.
Hypoxic skin injuries are also an important pathological process in many diseases, including multiple types of ulcer such as pressure, diabetic and varicose ulcers. Insufficient blood or oxygen supply is a leading causal factor, and can lead to chronic, non-healing ulcers. It has been shown that oxidative stress, the overproduction of reactive oxygen species (ROS), is intimately associated with hypoxic injury of skin. This has led to additional studies which have examined the potential protective ability of antioxidants against hypoxia and its downstream effects.
Here we demonstrate an in vitro multiplexed microplate assay that can monitor induction of hypoxia and oxidative stress through ROS production. Cobalt Chloride (CoCl2), a well-known mimetic agent of hypoxia, was used to chemically induce these phenotypes in immortalized keratinocytes. Relative whole well fluorescence intensity data were acquired for both assays using PMT-based fluorescence microplate detection, in addition to using automated digital wide-field fluorescence microscopy. The latter provides quantitative ratios of affected cells relative to the total cell population using the cellular analysis feature of Gen5™ software in addition to qualitative visual confi rmation of oxidative stress and hypoxia.
A screen of documented antioxidant compounds was performed to determine whether induction of the hypoxic condition could be inhibited using the cell model included here. A hit picking feature of the instrument’s software allowed for rapid screening of compounds using whole well intensities followed by imaging of only hit wells to collect phenotypic data. Dose response tests were then performed in order to confirm the effects seen from select inhibitor compounds. Finally, a luminescent cell viability assay was incorporated where cell viability measurements were performed in the same well following detection of the fluorescent signals from the Hypoxia/Oxidative Stress assay. All measurements were performed on the Cytation3 Cell Imaging Multi-Mode Reader.