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 use