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 Hsp90β 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 2. Hsp60, Hsp90β 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 Hsp90β (ltop panel). When detecting only Hsp60, the DyLight 549 (orange) Antibody is used (bottom panel).
Figure 3. Chemical induction of Hsp60 and Hsp90β in A549 cells. Different chemicals (top panel: CuSO4 bottom panel: menadione and ZnCl2) were added to cells at the indicated concentrations. Each data point represents 16 wells from three separate plates. Hsp60 and Hsp90β 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.
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