Cellomics HCS Reagent Kit for quantification of nuclear DNA content and heat shock proteins 60 (Hsp60) and 90 beta (Hsp90b). | | | | | | Hsp60 | Hsp90b | | Measure three parameters in the same cells | Determine dose responses |
DescriptionThe Cellomics Hsp60 and Hsp90b Detection Kits are for simultaneous quantification of nuclear DNA, heat shock protein 60 (Hsp60) and heat shock protein 90b (Hsp90b). These kits allow direct measurements using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The DNA binding dye, DAPI, enables nuclear size and morphology determination and cell cycle phase identification by DNA content. The primary antibodies are specific for their targets with minimal cross-reactivity. Heat shock proteins (HSP) are essential for protein folding, protein synthesis, cellular stress defense and many other functions. Cellular stress increases the HSP levels in cells by transcriptional regulation through HSF-1, STAT1, ATF3 and c Jun. This cellular response is critical for cellular homeostasis. Induction of HSP is closely correlated with substance cytotoxicity and lipophilicity given to the cell. Hsp60 has several immunological effects, such as induction of pro-inflammatory cytokine secretion from a number of myeloid and vascular cell types (e.g., smooth muscle cells). Hsp60 is induced with the mitochondria-specific stress response in mammalian cells in addition to heat stress. Cytosolic Hsp60 binds to Bax and Bak, blocking their pro-apoptotic ability. Hsp60 protects mitochondrial functions after ischemic injury in both cardiac and muscle cells and inhibits cell death. Hsp90 is one of the most abundant proteins in eukaryotic cells and has two isoforms, a and b. Hsp90 has chaperone activity with many other co-chaperones, which is important in folding of specific proteins of various signaling pathways. Because the altered chaperone function of HSP is associated with the development of many human diseases including cancer, neurodegenerative disorders, and cardiovascular disease, making chaperones targets for drug development. The Hsp60 and Hsp90b Detection Kits have been optimized with the Thermo Scientific ArrayScan® HCS Reader using the Compartmental Analysis BioApplication Software Module. Thus, automated plate-handling, focusing, cell image acquisition/processing, and data analysis/management 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. | | Figure 1. Hsp60, Hsp90b and nuclei staining in A549 cells. Cells were non-treated or treated with CuSO4 for 24 hours and stained as described in the protocol and imaged using the ArrayScan VTI HCS Reader. When detecting both targets, a DyLight 488 (green) Antibody is used for Hsp60 and a DyLight 549 (orange) Antibody is used for Hsp90b (left panel). When detecting only Hsp60, the DyLight 549 (orange) Antibody is used (right panel). |
| | Figure 3. Chemical induction of Hsp60 and Hsp90b in A549 cells. Different chemicals (left panel: CuSO4, right panel: menadione and ZnCl2) were added to cells at the indicated concentrations. Each data point represents 16 wells from three separate plates. Hsp60 and Hsp90b were measured using the output parameter of nuclear fluorescence with the Compartmental Analysis Bioapplication. Values were normalized with the maximum control value. EC50 values are listed. |
Related Poster Presentations | Suk J. Hong and Richik N. Ghosh (2008) Detection of Cellular Stress Responses to Drugs: Heat Shock Proteins are Powerful Cell Stress Indicators in Cell-Based, High-Content Assays. |
Kit Contents| Component | 8406701 | 8406702 | 8406801 | 8406802 | | Hsp60 Primary Antibody (mouse) | 25 µl | 120 µl | 25 µl | 125 µl | | Hsp90b Primary Antibody (rabbit) | 15 µl | 60 µl | -- | -- | DyLight 488 Conjugated
Goat Anti-Mouse IgG | 14 µl | 72 µl | -- | -- | DyLight 549 Conjugated
Goat Anti-Rabbit IgG | 14 µl | 72 µl | -- | -- | DyLight 549 Conjugated
Goat Anti-Mouse IgG | -- | -- | 30 µl | 72 µl | | DAPI Dye | 50 µl | 50 µl | 50 µl | 50 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | 100 ml | 100 ml | Permeabilization Buffer (10X D-PBS with 1% Triton® X-100) | 100 ml | 100 ml | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack | 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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