Cell Death Detection KitMultiparameter Cell Death Detection Kit
Last Updated on Friday, 04 November 2011 05:13
Overview
Cellomics HCS Assay for detection of four parameters relating to cell death.
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Nuclei |
Cytochrome C
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LC3B
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Measure four parameters in the same cells
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Determine dose responses
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The Thermo Scientific Cellomics Multiparameter Cell Death Detection Assay for high-content screening (HCS) enable simultaneous quantitation of LC3B protein on autophagic vesicles, cytochrome c localization and its release from mitochrondria, cell membrane integrity and its permeability and nuclear morphology and DNA content. These cellular properties are measured directly using fixed end-point assay and fluorescence detection in cells grown on standard high-density microplates. The Assay contains 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 Assay includes reagents with 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.
Features
The reagents have been optimized for use with the ArrayScan VTI HCS Reader, CellInsight Personal Image Cytometer 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 these reagents can be viewed and analyzed by fluorescence and confocal microscopes.
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Figure 1. 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). |
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Figure 2. 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 VTI HCS Reader. |
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Other drug response that can be graphed with these multiparameter cell death data:
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Figure 3. Dose response curve for cell permeability in HepG2 cells treated with staurosporine. |
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Ordering
Each assay offers all the components necessary to perform the assay as well as a validated protocol, with step-by-step instructions for use on any of the Thermo Scientific HCS Readers as well as other instrumentation and standard fluorescence microscopes. Reagents and Assays are available in kit form (everything in the box) or as components where based on your need, so you may purchase all or al-la-carte reagents for the assay.
To order Cellomics HCS Reagents, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.
To order components for a legacy kit please visit our ReagentFinder page
