| Jun
1, 2005 |
| By:
Vivek
C. Abraham, Jeffrey
R. Haskins |
| Pharmaceutical
Discovery |
|
We have developed a high-content bioassay that enables fully
automated measurement of oxidative stress in individual cells in
microplate format. Here we report the automation of this bioassay, which
quantitatively measures cytotoxicity in terms of the rate and magnitude of
intracellular generation of reactive oxygen species.
Introduction High-content
screening (HCS), or the automation of image-based cellular investigation,
rapidly is being adopted for large-scale performance of bioassays
involving manipulation of several experimental variables. Briefly, HCS
involves seamless automation of image acquisition, image analysis (BioApplications)
and data extraction, data viewing/analysis and data archival. HCS
applications include cytotoxicity screening of compounds for safety
assessment and/or lead optimization and detailed investigation of
mechanisms of action.
Reactive oxygen species (ROS) generation is known to play a role in
several pathological conditions including ischemia, neurodegeneration and
vascular damage. Monitoring changes in the rate of ROS production is
routinely possible through the use of a variety of fluorescent probes.
Here, we focus on the automation of a bioassay that uses dihydroethidium (DHE),
a widely used fluorescent indicator for ROS detection. Intracellular
oxidative factors, such as superoxide ions and/or hydroxyl radicals, can
oxidize non-fluorescent DHE to fluorescent ethidium (1, 2). The formation
of ethidium, a DNA intercalator, can be readily monitored by measuring its
accumulation within the cell nucleus. In addition, imaging-based
approaches also can be used to simultaneously monitor other cellular
indicators of toxicity, such as whole-cell and/or nuclear morphology and
plasma membrane integrity.
Figure 1. Menadione-induced
oxidative stress is seen in primary rat cortical neurons.
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Experimental Conditions The assay for
measurement of ROS generation was implemented with cryopreserved primary
rat cortical neurons (Cambrex Corp., East Rutherford, NJ, USA) in 96-well
microplates. Cells were cultured continuously for five days to reduce the
subpopulation of non-neuronal cells, as per the vendor's recommendations.
Cells then were incubated in Cellomics' (Pittsburgh, PA, USA) KineticScan®
HCS Reader (KSR) with culture medium containing Hoechst 33342 (Molecular
Probes) to enable identification of cell nuclei. A green fluorescent cell
permeability indicator also was added to label and gate out necrotic
cells. Freshly prepared solutions of DHE (final concentration = 1 µM)
with varying concentrations of menadione (a redox cycling quinone) in cell
culture medium then were added to the cells. This was immediately followed
by fully automated monitoring of the kinetics of ROS generation through
use of the Target Activation BioApplication on the KSR.
Figure 2. Population-averaged
response of primary rat cortical neurons to menadione exposure is
shown.
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Results Figure 1 shows ROS generation
in rat cortical neurons by exposure to 100 μM menadione, indicated by
increase in the content of fluorescent ethidium:DNA complex in individual
cells. A green fluorescent cell permeability indicator was used to label
and gate out nuclei/nuclear debris associated with necrotic cells having
compromised membrane integrity. This was done to eliminate erroneous
calculation of the rate of DHE oxidation from cells with little or reduced
metabolic capability. Figure 2 shows the results of automated quantitation
of ROS generation. Error bars represent the standard deviation across four
replicate wells for each dose. Significant dose-related increases in the
rate of DHE oxidation are seen as early as 40 minutes post exposure.
Oxidation rates within the first 40 minutes were 3.2, 5.1 and 8.2
fluorescence units/min. for menadione exposures of 2, 100 and 500 μM,
respectively. At later time points, exposure to 500 μM menadione
displayed a reduced rate of DHE oxidation, presumably due to the cells'
metabolic machinery being severely damaged by exposure.
Conclusions
We have demonstrated the application of HCS to the automation of a kinetic
bioassay for measurement of the rate and/or magnitude of ROS generation in
neurons. Our results provide proof of principle for implementation of this
bioassay as a component of large-scale experimentation involving the
profiling of oxidative stress responses as a function of several
experimental variables.
References
1. V.P. Bindokas, J. Jordan, C.C. Lee and R.J. Miller, J Neurosci. 16,
1324–36 (1996).
2. T. Münzel, I.B. Afanas'ev, A.L.
Kleschyov and D.G. Harrison, Arterioscler Thromb. Vasc. Biol. 22,
1761–1768 (2002).
Cellomics Inc. 100
Technology Dr., Pittsburgh, PA 15219 USA
Tel. 412-770-2200; Fax 412-770-2450
www.cellomics.com.
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