Cellomics HCS Reagent Kit for simultaneous analysis of three cell cycle parameters: DNA content, BrdU incorporation and phospho-Histone H3 activation. | | | | | | BrdU
Incorporation | Histone H3
Activation | Analyze | Conclude |
DescriptionThe Cellomics Cell Cycle Kit I – BrdU and Phospho-Histone H3 Kit is for simultaneous quantification of nuclear DNA content to distinguish 2N (G1 phase) and 4N (G2/M phase), DNA replication in S phase cells, and mitosis marker in M phase cells. These kits allow direct measurements of BrdU incorporation and mitosis-specific histone H3 phosphorylation using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The DNA binding dye DAPI is used to determine the cell cycle phases by DNA content. The primary antibodies are specific for their targets and have minimal cross-reactivity with other targets. The Cellomics Cell Cycle Kit I detects BrdU and phospho-Histone H3 in nuclear DNA without harsh treatments, enabling multiplexed detection of BrdU with antibodies toward other cellular targets. This assay is sensitive, rapid and easy to use. The evaluation of DNA replication, mitosis and other information such as cell number, the state of cell cycle-associated proteins, morphology and analysis of other cellular antigens can be obtained from a single culture. These kits have been optimized with the Cellomics ArrayScan HCS Reader using the Target Activation BioApplication Software Module. Thus, automated plate-handling, focusing, cell image acquisition and processing, and data analysis are combined in one high-content screening (HCS) system to assay for test compounds. In addition to HCS instruments, cells labeled by the kit reagents can be viewed and analyzed by fluorescence and confocal microscopes. B.
A. C.
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| Figure 1. BrdU and phospho-Histone H3 staining with cell cycle inhibitory drugs. A549 cells were treated with hydroxyurea (10 mM), nocodazole (0.5 µg/ml), vinblastine (0.3 µM), cycloheximide (10 µg/ml), mimosine (0.5 mM) and sodium butyrate (5 mM) for 24 hours and stained with BrdU and phospho-Histone H3 antibodies as described in the protocol. Panel A: Images of nuclei, BrdU and phoshpo-Histone H3 in drug-treated A549 cells. Panel B: Activation or inhibition of BrdU and phospho-histone H3 after drug treatment for 24 hours. Panel C: Cell cycle drugs were added to the cells at the indicated concentrations. BrdU was measured using the output parameter of the nuclear fluorescence total intensity (MEAN_TotalIntenCh2) and phospho-histone H3 was measured using the nuclear fluorescence average intensity (MEAN_AvgIntenCh3) with the Cellomics Compartmental Analysis Bioapplication. Values are normalized with the maximum control value and presented as percent maximum in Panel B and percent control in Panel C. |
Related Poster Presentations | Suk J. Hong and Richik N. Ghosh (2008) Quantitative Analysis of Drug Effects on Cytoskeletal Structure, Cell Morphology and the Cell Cycle by High-Content Assays. |
Kit Contents| Component | 8404601 | 8404602 | | BrdU | 100 µl | 100 µl | | BrdU Primary Antibody (mouse) | 110 µl | 600 µl | | Phospho-Histone H3 Primary Antibody (rabbit) | 8 µl | 30 µl | | DyLight 488 Conjugated Goat Anti-Mouse IgG | 14 µl | 72 µl | | DyLight 549 Conjugated Goat Anti-Rabbit IgG | 14 µl | 72 µl | | MgCl2 | 500 µl | 500 µl | | DAPI Dye | 50 µl | 50 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | Permeabilization Buffer (10X D-PBS with 1% Triton® X-100) | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack |
Ordering InformationScreening size kits and components (bulk and custom) available with special pricing. Please call. To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products. Choose "Order Online" in table to see pricing and order online (U.S. only). See all Cellomics HCS Reagent Kits
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