( washed and imaged. For time course expe...)Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays

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.

Results

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.

Discussion

Activation of the caspase-3 pathway is a hallmark of apoptosis and can be use
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