Key words: In Cell Analyzer • apoptosis • multiplexing • cell based assays
Apoptosis represents a gene-directed, morphologically and biochemically distinct form of programmed cell death. Inappropriate control of apoptosis is associated with a variety of disease states such as cancer, AIDS, neurodegenerative diseases, and ischaemic stroke. Hence, the mechanism and control of apoptosis is extremely important when considering the effects of new drugs on the body, or when targeting the process itself for therapeutic purposes.
The IN Cell Analyzer 3000, a high-throughput confocal imaging system, provides a means of real time, nondestructive quantitation of morphological and biochemical cellular changes. Several different endpoints can be monitored simultaneously on a cell-by-cell basis or by using a population average. Compared to more traditional single-plex techniques, multiplexed data acquisition increases the reliability of the information because data from multiple assays are derived from the same population of cells. This has the added benefit of making the most of scarce tissue/cell samples. Samples can be analyzed in a real-time or off-line mode using robust and flexible analysis modules.
In this application note, two assays are multiplexed to monitor cytotoxicity in primary rat hepatocytes. After exposure to known inducers of apoptosis, IN Cell Analyzer 3000 is used to acquire images and quantitate changes in nuclear and mitochondrial status. Primary rat hepatocytes are exposed to staurosporine or diclofenac for 4- and 21-h respectively. Staurosporine is a microbial alkaloid, a strong inhibitor of protein kinases, thought to work through JNK1, caspase proteases, AP-1, and NFκβ. It is an apoptotic inducer causing nuclear condensation (1, 2). Diclofenac is a non-steroidal anti-inflammatory compound that has been associated with liver damage and covalent protein-adduct formation. A toxic metabolite formed from diclofenac by the action of the cytochrome P450 system has been reported to induce apoptosis with nuclear condensation (3).
IN Cell Analyzer 3000 25-8010-11
Granularity Analysis Module
(for IN Cell Analyzer 3000) 63-0048-97
Other materials required
Primary rat liver cells (Bowman Research UK)
96-well, black, flat-bottom microtiter plates (Packard View Plate, Packard).
Hoechst 33342 (Molecular Probes)
MitoTracker™ Deep Red 633 (Molecular Probes)
1:1 mix of Williams’ E and Hams F-12 with additions of 5 µg/ml Insulin,
5 µg/ml Transferrin, 5 µg /ml Selenium, 10 nM Dexamethasone, 2 % v/v fetal calf serum,
10 mM HEPES, 50 U/ml Penicillin G, 50 µg/mlStreptomycin sulfate.
All tissue culture reagents were obtained from Gibco BRL, UK unless otherwise stated.
Fetal bovine serum (Sigma)
Phosphate buffered saline (PBS) solution
Alamar Blue™ (Serotec)
1. Plate out one set of freshly isolated primary rat hepatocytes in growth media containing 0 µM, 50 µM, 150 µM, or 300 µM diclofenac in a total volume of 200 ml in a 96-well plate for 21 h. The seeding density is 20 000 cells per well. Cells are acceptable at a minimum of 80% viability immediately prior to culture at a maximum of 1 h post isolation. The wells containing no compound are taken as controls.
2. Plate out a second set of primary rat hepatocytes in 200 ml growth media for 21 h prior to dosing with staurosporine.
3. Following the 21 h adherence period, dose the second set of primary rat hepatocytes with 0 µM, 0.5 µM, 1 µM, or 5 µM staurosporine respectively for 4 h in growth media. The wells containing no compound are taken as controls.
4. After the appropriate exposure period, remove the dosing media and replace it with media containing MitoTracker Deep Red 633 (25 µM) and Hoechst 33342 (1 µM) probes in a final volume of 100 µl per well for 10 min at 37 ºC, 5% CO2.
5. Image on the IN Cell Analyzer 3000 at 37 ºC using the following filter sets: Hoechst Ex. 352 nm, Em. 455 nm; MitoTracker Deep Red 633 Ex. 644 nm, Em. 665 nm.
6.Analyze the images using the granularity algorithm of the IN Cell Analyzer 3000 in both the nuclear and cytoplasmic compartments.
The protocol provided allows nuclear changes that occur during apoptosis to be tracked using the DNA binding dye Hoechst 33342. Non-apoptotic cells exhibit homogeneous nuclear staining (Fig 1A). As cytotoxicity occurs with increasing dose of compound, the staining reveals an increasingly textured punctate pattern (Fig 1B) attributable to nuclear DNA condensation. Mitochondrial changes are monitored using MitoTtracker Deep Red 633. The observed morphology changes from an intense punctate distribution of the dye (Fig 1D) to a dimmer, more diffuse pattern (Fig 1E) with increasing concentration of compound .
The Granularity Analysis Module is applied sequentially to analyze the nuclear and cytoplasmic staining. This module automatically defines measurement areas of interest such as nuclei and cell bodies (Figs 1C and 1D), and then quantitates qualifying ‘granules’ of a specified dimension detected within the measurement region. For both assays, the nuclear image is used as a starting point to define the measurement region, which can be adjusted to encompass either the entire cell body or just the nuclear area. A granule size is then chosen to best represent the biology and the punctate structures are automatically quantitated in the desired channel (blue channel for nuclear measurements; red channel for mitochondrial measurements).
During development and validation of the protocol, four additional more traditional cell-based assays were run in parallel to determine the cytotoxicity of diclofenac in primary rat hepatocytes. The assay giving the lowest EC50 value with the most reproducible data, Alamar Blue, was then taken as the benchmark assay to compare the sensitivity of the nuclear and mitochondrial status assays (1). Alamar Blue is a tetrazolium salt sensitive to cellular redox levels and is widely used to assess cytotoxicity.
For all data plots shown, the results are expressed as a percentage of control values obtained from cultures receiving no test compound. For the mitochondrial and nuclear status assays, the percentage response is calculated from the population-averaged Ngrain value, Ngrains being the number of grains detected per cell or per nucleus (depending on the assay). The Granularity Analysis Module is also used to determine cell number per image.
Following exposure to staurosporine or diclofenac, primary rat hepatocytes show early signs of cytotoxicity. DNA condensation is clearly observed in treated cells compared to controls (Figs 2 and 3). There is also a distinct decrease in mitochondrial punctate fluorescence following treatment (Figs 1D and 1E). This decrease reflects changes in mitochondrial permeability that occur during apoptosis and necrosis. U sing the IN Cell Analyzer 3000 system and software it is possible to detect and quantitate these changes.
Cells exposed to staurosporine for 4 h are viable at all exposure concentrations as shown by the Alamar Blue assay data (Fig 4). Even at the highest dose, there is no significant change in Alamar Blue signal relative to controls. The cell number also remains fairly constant, dropping to 76% of controls only at the highest dose of staurosporine. However, the nuclear measurements show concentration-dependent changes in nuclear texture consistent with DNA condensation (Fig 4). Dose-dependent decreases in mitochondrial staining with MitoTracker Deep Red 633 are also observed. Thus, although little or no toxicity is detected using traditional measurements such as cell number and Alamar Blue, the nuclear and mitochondrial measurements obtained using IN Cell Analyzer 3000 clearly indicate that staurosporine-induced cell death is well underway.
Similar to staurosporine treatment, diclofenac treatment results in little change in the Alamar Blue data; cells are viable at all concentrations of diclofenac after 21 h of exposure (Fig 5). Cell numbers do not deviate statistically from control values at any of the compound concentrations. DNA condensation; however, increases at the highest dose of diclofenac to 240 % of control values, while punctate mitochondrial staining decreases at the highest dose of diclofenac to less than 60% of control values. Indications from the Alamar Blue reference assay and cell number measurements are that there is no overt toxicity occurring in the system at any dose of diclofenac. By contrast, it is clear from the data acquired with the IN Cell Analyzer 3000 system that cytotoxicity can be detected at 300 mM diclofenac.
The IN Cell Analyzer 3000 system and software can be used successfully to quantitate quantify changes in nuclear and mitochondrial status following treatment with apoptosis-inducing agents such as staurosporine and diclofenac. Assays assessing nuclear and mitochondrial status can be multiplexed, thereby reducing the quantities of primary cells and compounds required. Rapid, sequential data analysis allows the results for each of the multiplexed assays to be generated in approximately 0.2 s per image, representing a significant time-savings over more traditional methods, and thereby enabling high-throughput assessment of cytotoxicity.
Nuclear DNA condensation, typical of late stage apoptosis, and mitochondrial changes are clearly visible and can be quantitated at the lowest concentration of staurosporine (0.5 µM) and at 300 µM diclofenac. By contrast, the Alamar Blue assay is not capable of detecting cytotoxic effects relative to controls even at the highest concentrations of these compounds. Thus, DNA condensation and mitochondrial changes are more sensitive indicators of cytotoxicity than the conventional Alamar Blue assay.
Multiplexing DNA condensation and mitochondrial status may be useful in determining mechanisms of drug action. In addition, the multiplex format has the advantage that each assay is performed on the same population of cells, thereby increasing confidence in the correlation between endpoints.
1. Feng, G. and Kaplowitz, N. Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am J Physiol Gastrointest Liver Physiol. 282(5), G825–34 (2002).
2. Sanchez, V. et al. Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes. Biosci Rep. 12(3), 199 –206 (1992).
3. Gomez-Lechon, M.J. et al. Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochem Pharmacol. 66(11), 2155–67 (2003).
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