These kits allow direct in-cell measurements using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The primary antibody is specific for human cleaved caspase 9 and does not cross-react with total caspase 9 or other caspases. The secondary antibody is conjugated to DyLight™ 549 Fluor (orange).
The kit reagents have been optimized for use with the ArrayScan® HCS Reader and the Target Activation BioApplication Software Module, but they also can be used with other Cellomics BioApplication Modules. Thus, automated plate-handling, focusing, cell image acquisition/processing, and data analysis and management have been combined into a high-content screening (HCS) system to assay for test compounds affecting the activation of caspase 9. In addition to HCS instruments, cells labeled by the reagents in this kit can be viewed and analyzed by other fluorescence microscopes.
Figure 1. Staining of cleaved (active) caspase 9 in HeLa cells treated with vehicle (0.1% DMSO in media) or with 1 µM staurosporine for 4 hours. Cells were stained according to the kit protocol and imaged using a Cellomics ArrayScan HCS Reader. Cells treated with stautosporine results in caspase 9 activation and an increase in staining.
Figure 2. Caspase 9 activation in HeLa cells. The assay was performed as described in the procedure using different doses of staurosporine treatment for 4 hours. The feature plotted is the percent of cells that are responders for caspase 9. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of staurosporine).
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