The understanding of the cellular signaling processes leading to programmed cell death (apoptosis) is of utmost importance in the study of autoimmune diseases such as rheumatoid arthritis or Parkinsons and Alzheimers disease. Here, errors in the signaling cascade are believed to lead to premature apoptosis in the affected tissue. At the same time, damaged cells that do not enter the apoptotic pathway may proliferate unchecked and become a cancerous cell mass. Thus, the study of apoptosis benefits many different fields of medicine by offering multiple targets for specific drug treatment.
Caspases (cysteinyl aspartate-specific proteases) are a family of important signaling molecules with various tasks depending on subtype and organ. The activation of caspases is also a marker for cellular damage in diseases such as stroke or myocardial infarction (reviewed by Lavrik et al . 2005). Although the precise role in the initiation and progression of apoptosis is not known for all caspases, their involvement as an indicator alone and as a potential leverage point for drug treatment make them widely researched molecules.
We explored the use of the active form of caspase-3 for the detection of apoptotic events. This protease has been implicated as an effector caspase associated with the initiation of the death cascade and is therefore an important marker of the cells entry point into the apoptotic signaling pathway (Nicholson et al . 1995). Caspase-3 is activated by the upstream caspase-8 and caspase-9 and since it serves as a convergence point for different signaling pathways, it is well suited as a read-out in an apoptosis assay. Detection of active caspase-3 can be used in different cell lines or primary cells, does not require the use of transfection techniques and can be multiplexed with other probes to get an in-depth understanding of signaling events with cell-by-cell resolution. Since there have been reports that cells progressively undergo apoptosis when exposed to staurosprine over time (Jacobson et al . 1996), we investigated the time-dependence of apoptosis as measured by caspase- 3 activation. This application is also amenable for use in automated microscopes such as the BD Pathway Bioimager in a hands-off mode, making it useful for secondary screening applications or to gain an insight into apoptotic pathways using RNAi.
HeLa cells (ATCC, CCL-2, a human cervical cancer cell line) were maintained and grown in DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum. Cells were seeded in 96-well clear bottom tissue culture plates optimized for imaging applications (Cat. No. 353219) at 10,000 cells/well. After 16 hours in the incubator, staurosporine was added to the cells for 4 hours.
Staurosporine (Sigma-Aldrich, S4400) is a broad-spectrum kinase inhibitor known to activate the apoptosis pathway. To generate dose-response relationships, the drug was serially diluted in growth medium and equal volumes of a 2 final concentration was added to the wells; the final vehicle (DMSO) concentration did not exceed 0.5%. After incubation, cells were fixed with 3.7% formaldehyde (in PBS) for 1h at room temperature and methanol (100%) permeabilized for 10 min at 20C. Non-specific binding was blocked for 1h with 5% goat serum in PBS buffer containing 0.3% Triton X-100. Cells were then incubated with cleaved caspase-3 (Asp 175) rabbit polyclonal antibody (Cell Signaling Technology, 9661, 1:100 dilution) in 5% goat serum and 0.3% Triton X-100 over night at 4C. Further wash steps removed unbound antibody prior to the addition of the secondary Alexa 488 goat anti-rabbit antibody (Invitrogen, A11034, 10μL/mL) and Hoechst 33342 dye (5 μg/ml) in 5% goat serum and 0.3% triton X-100 for 1 hr at room temperature. The cells were then washed and imaged. For time course experiments, the drug treatment was staggered in hourly intervals and cells were fixed at the end of the experiment.
Images were acquired on the BD Pathway Biomager using the 20/NA 0.75 objective. The system allows great flexibility in the selection of the fluorescent dyes since it uses 2 lamps with broad illumination spectrum and a total choice of 16 excitation filters. Hoechst 33342 (Invitrogen, H3570) and Alexa Fluor images were acquired using standard filter sets for dyes in this range with careful consideration of bleed-through artifacts. The BD Pathway Bioimager also has sophisticated analysis software built in to acquire and analyze images with minimal manual adjustment. Caspase-3 is primarily located in the cytoplasm and the algorithm was set to sample an area around the nucleus as representative of the cytoplasm.
Images acquired by the BD Pathway Bioimager showed a distinct labeling pattern of the activated caspase-3 in response to the apoptosis-inducing drug staurosporine ( Figure 1 ). The doseresponse relationship is highlighted in Figure 1 showing a screen capture of the acquisition software. Each thumbnail image shows a representative area of the sampled well.
The entry into the apoptotic pathway is terminal and once a cell commits, the caspase-3 pathway is completely activated. Therefore, the best measure of the relative apoptotic activity in a well is the percentage of cells above a certain threshold. The BD Image Data Explorer data analysis tool allows setting of multiple thresholds and to plot dose-response curves, calculate EC50 ( Figure 3 ) and Z using these percentages.
Activation of the caspase-3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify activators and inhibitors of the death cascade. The response is both time and concentration dependent suggesting that multiple pathways play a role in triggering the caspase-3 activation. One could hypothesize that cells are most susceptible to staurosporine in a specific phase of the cell cycle and therefore over time, most cells will die, similar to the findings of Vermeulen et. al. (2003).
We show that this representative apoptosis assay can be used in an automated way using the BD Pathway Bioimager and its built-in image and data analysis tools making it amenable to a high-throughput application development. Our data supports findings by Jessel et. al. (2002) who report an EC50 for staurosporine of 200 nM. We specifically see the usefulness of this assay as a building block in a multiplexed high-content assay. This caspase-3 assay can easily be accommodated in a different fluorescence channel and since the BD Pathway Bioimager utilizes broad-spectrum lamps, many dye combinations are possible. The BD Pathway Bioimager and its software tools provide the optimal environment to conduct this kind of multidimensional analysis.
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