The protocol is performed on live cells growing on standard high-density microplates, and detection is accomplished via immunofluorescence. The kit includes a primary STAT3 antibody and a DyLight™ 488-conjugated Secondary Antibody. The nuclear region is identified by Hoechst Dye, which is also included.
Cytokines are important for the proliferation and differentiation of hematopoietic cells. Many cells respond to cytokine induction via the janus kinases-signal transducers and activators of transcription (JAK/STAT) pathway, resulting in the transcription of selected genes. The complement of induced genes and associated response varies according to the cell type and stimulus, which is reflected by tyrosine phosphorylation by specific JAKs. Consequently, it is crucial that screens for potential drugs that affect this collection of pathways consider specificity among the six known STAT targets. This specificity is afforded by this HCS reagent kit, where activation of STAT3 is quantified by its translocation to the nucleus.
The optimized protocol included in the STAT3 Activation HCS Reagent Kit product is for a fixed end-point assay. Inhibitors of STAT3 translocation are screened by stimulating cells with a control inducer such as interferon-α (IFN-α) after exposure of live cells to test compounds. Replacing IFNα in the assay with test compounds identifies agonists of STAT3 translocation. Translocation is directly quantified as the difference in cytoplasmic to nuclear intensity of the labeled transcription factor. The STAT3 Activation HCS Reagent Kit, in combination with the Cellomics ArrayScan® HCS Reader and the Cytoplasm to Nucleus Translocation Application software, enables automated plate handling, focusing, cell image acquisition, analysis and quantification of STAT3 activation. For a more detailed description of the image processing algorithm, see the Cytoplasm to Nucleus Translocation Application Guide that accompanies the Cytoplasm to Nucleus Translocation Application software.
Figure 1. HeLa cells stained before and after STAT3 activation by IFN-α. Top panels: STAT3 localization in non-treated cells. Bottom panels: STAT3 localization in stimulated cells (IFN-α – 2500 U/ml for 20 minutes).
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