Cellomics HCS Reagent Kit for quantification of nuclear DNA content and heat shock proteins 70 (Hsp70) and 90 alpha (Hsp90a). | | | | | | Hsp70 | Hsp90a | | Measure three parameters in the same cells | Determine dose responses in cytoplasm and nuclei |
DescriptionThe Cellomics Hsp70 and Hsp90a Detection Kits are for simultaneous quantification of nuclear DNA content, heat shock protein 70 (Hsp70) and heat shock protein 90a (Hsp90a). 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 and have 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, which is critical to maintain cellular homeostasis. Induction of HSP is closely correlated with substance cytotoxicity and lipophilicity given to the cell. Hsp70 is involved in guiding protein translocation across organelle membranes, disassembling oligomeric protein structures, facilitating proteolytic degradation of unstable proteins and controlling the biological activity of folded regulatory proteins. Nuclear localization of Hsp70 is associated with cell stress and cell growth. Hsp90 is one of the most abundant proteins in eukaryotic cells. Hsp90 has chaperone activity with many other co-chaperones and is important for folding proteins of various signaling pathways. The two isoforms of Hsp90, alpha and beta, have the same function. 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 Hsp70 and Hsp90a 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 and processing, and data analysis and 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. Hsp70, Hsp90a and nuclei staining in A549 cells (20X objective). Cells were non-treated or treated with CuSO4 (1 mM) for 24 hours, stained as described in the protocol and imaged using the ArrayScan VTI HCS Reader. |
| | Figure 2. Chemical induction of Hsp70 and Hsp90a in A549 cells. Cells were treated with different concentrations of chemicals (left panel: CuSO4, right panel: menadione and ZnCl2). Each data point represents 16 wells from three separate plates. Hsp70 and Hsp90a were measured using the output parameter of nuclear fluorescence or the difference between cytoplasm and nucleus average fluorescence intensity (cyt-nuc diff) with Cellomics 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 | 8406301 | 8406302 | 8406401 | 8406402 | 8406501 | 8406502 | | Hsp70 Primary Antibody (mouse) | 12 µl | 60 µl | 12 µl | 60 µl | -- | -- | | Hsp90a Primary Antibody (rabbit) | 15 µl | 60 µl | -- | -- | 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 | -- | -- | 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 | 50 µl | 50 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | 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 | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | 85 ml | 85 ml | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack | 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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