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Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays

l 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.

Methods

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
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