Multiparameter Cell Death Detection Kit

These cellular properties are measured directly using fixed end-point assay and fluorescence detection in cells grown on standard high-density microplates. The kits contain highly specific primary antibodies, DyLight™-conjugated Secondary Antibodies and other reagents and buffers required to stain cells for HCS assays.

Programmed cell death is characterized by morphological changes that include classical apoptotic features such as loss of mitochondrial membrane potential, increased cell permeability, induction of nuclear condensation and fragmentation. Mitochondrial-dependent apoptosis is accomplished by release of cytochrome c and cleavage of caspase 9, caspase 3 and PARP. The apoptosis mechanism is mainly caspase-dependent; however, other forms of programmed cell death, such as that induced by autophagy, is caspase-independent. Autophagy is a catabolic process involving sequestration and self-ingestion of various cellular constituents such as organelles and long-lived proteins. LC3B is one of the best known markers that indicate formation of autophagic vesicles. Cells that undergo excessive autophagy are triggered to die in a non-apoptotic manner. Automated, quantitative, cell-based imaging HCS assays enable simultaneous measurements of multiple targets in individual cells, allowing programmed cell death pathways to be distinguished and characterized. This kit has markers for apoptosis (cytochrome c), autophagy (LC3B), general cytotoxicity and cell death indicators (nuclear morphology, DNA content and cell membrane permeability), which are monitored in the cells simultaneously.

This kit was optimized using the Thermo Scientific ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module but can be used with other Cellomics BioApplications. Thus, automated plate-handling, focusing, cell image acquisition and data analysis are combined in one HCS system to assay for compounds that regulate the autophagy and other apoptotic events. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.

Figure 2. Staining of nucleus (DAPI), cytochrome c and LC3B in HepG2 cells treated with media (non-treated) or with the proteasome inhibitor MG132 (20 µM) for 18 hours. Note that LC3B appears as distinct spots in the cytoplasm. Cytochrome c released from mitochondria is detected in cytoplasm and in the nucleus. Images from two time points were chosen to depict events that occur in their maximal states. LC3B expression (autophagy) and cytochrome c release from mitochondria and its distribution throughout the cell (this figure) precede nuclear condensation, loss of cells and increased cell permeability and terminal events associated with cell death (next figure).
 

Figure 3. DAPI (nuclear stain) and cell permeability dye staining of HepG2 cells treated with media (non-treated) or with the proteasome inhibitor MG132 (20 µM) for 28 hours. Enlarged images (3X) to the right illustrate MG132 drug exposed cells stained with DAPI (nuclear staining) and cell permeability dye. Cells were stained according to the kit protocol and imaged using a 10X objective on the ArrayScan V^TI HCS Reader.

Figure 4. Dose response curve for cell permeability in HepG2 cells treated with staurosporine.  Other drug response that can be graphed with these multiparameter cell death data:

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