Cellomics HCS Reagent Kit for detection of cyclin B1 and analysis of cell cycle phases.

detect cyclin B1	measure DNA content	analyze	characterize cell cycle	calculate dose response

Description

The Cyclin B1 Activation Kit enables measurement of intracellular cyclin B1 and its translocation to the nucleus during the different cell cycle phases. The kit contains a monoclonal anti-cyclin B1 mouse primary antibody, a goat anti-mouse secondary antibody conjugated to DyLight 549 Fluorophore, DAPI to identify cell-cycle phase by DNA content and various other reagents and buffers required for immunofluorescence staining of cyclin B1 for high-content screening (HCS) assays.

The activation of different cyclin/Cdk complexes is tightly controlled throughout the cell cycle. Among different cyclin/Cdk complexes, cyclin B1 in cooperation with Cdc2 (Cdk1) plays essential roles in mitosis in eukaryotic cells. Cyclin B1 binds to Cdc2 (Cdk1) serine/threonine kinase and the activated cyclin B1/Cdc2 (Cdk1) heterodimer induces mitosis by phosphorylating proteins that are involved in chromatin condensation, nuclear membrane breakdown and mitotic microtubule reorganization. Cdc2 (Cdk1) is phosphorylated by Wee1 and Myt1 at Thr14 and Tyr15, which deactivates Cdc2 (Cdk1). In contrast, dual specificity phosphatase Cdc25 dephosphorylates Cdc2 (Cdk1) and activates cyclin B1/Cdc2 (Cdk1) complex, which promotes mitosis. G2 to M phase (mitosis) transition is controlled by the M-phase promoting factor (MPF) and cyclin B1/Cdc2 (Cdk1) complex is the regulatory subunit of MPF in mammalian cells. The activity of cyclin B1 is closely associated with its intracellular level and subcellular distribution. The activation of cyclin B1 can be monitored by the intensity change of cyclin B1 during the cell cycle as well as by its translocation from the cytoplasm to the nucleus in prophase (Figure 1). Cyclin B1 is also the target of multiple mitotic checkpoints with DNA damage.

This kit was optimized using the Cellomics 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 activate cyclin B1. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.

DNA Content (DAPI)	Cyclin B1 (orange fluor)	Merged Image (DNA=blue, cyclinB1=green)
Figure 1. Cyclin B1 staining in asynchronous A549 lung carcinoma cells. Asynchronous A549 cells were fixed and stained with anti-Cyclin B1 Primary Antibody and DyLight 549 Conjugated Secondary Antibody (center panel). Cells were also labeled with DAPI Dye for nuclear staining (left panel). The intracellular level of cyclin B1 increases during the progression of cell cycle and cyclin B1 moves from the cytoplasm to the nucleus in mitotic cells. The cell cycle phases are determined from the DNA content analysis by DAPI staining, and the arrows show the change in intracellular cyclin B1 staining for the different cell cycle phases. Merged color image (right panel) is the same cell image with DAPI and DyLight 549 channels rendered blue and green, respectively.

A549 cells or IMR-90 cells were stimulated for 16 hours with different compounds to measure dose response of cyclin B1 (Figure 2). Images are of representative wells treated with nocodazole. The dose response curves were generated using different drug treatments.

Figure 2. Drugs blocking G2/M phase induce cyclin B1 translocation to the nucleus. Different drugs were added to the cells at the indicated concentrations. Each data point represents eight wells from one to three separate plates. Cyclin B1 was measured using the output parameter of the cytoplasm and nuclear fluorescence average intensity difference (MEAN_CircRingAvgIntDiffCh2) with the Cellomics Compartmental Analysis Bioapplication. Values are normalized with the maximum control value and presented as percent control. Nocodazole treatment (0.5 µg/ml, 16 hours) increases the cyclin B1 intensity and DNA content in the nucleus of A549 cells.

Related Poster Presentations

	Bhaskar S. Mandavilli, Suk J. Hong, Krishna M. Vattem, and Richik N. Ghosh (2008) Quantitative, Cell-Based, High-Content Screening Assays for DNA Damage-Induced Cell Cycle Checkpoints.
	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

Ordering Information

Screening 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.

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