STAT1 Activation Kit

The kit provides a fixed end-point assay based on immunofluorescence detection of STAT1 in cells growing on standard high-density microplates. The core reagents supplied include a primary antibody specific for STAT1, and a DyLight™ 488-Conjugated Secondary Antibody. The cell nucleus is identified by DNA-specific Hoechst Dye, which is also included in the kit.

Cytokines are important for 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. Screens for potential drugs that affect this collection of pathways must consider specificity among the STAT targets. The STAT1 Activation Kit quantifies STAT1 by its translocation to the nucleus.

The optimized protocol included is for a fixed end-point assay. Inhibitors of STAT1 translocation are screened by stimulating cells with a control inducer such as interferon-γ (IFN-γ) after exposing live cells to the test compounds. Replacing IFN-γ in the assay with test compounds identifies agonists of STAT1 translocation. Translocation is directly quantified as the difference in cytoplasmic to nuclear intensity of the labeled transcription factor. The kit reagents, in combination with the ArrayScan HCS System and the Cytoplasm to Nucleus Translocation Application software enable automated plate handling, focusing, cell image acquisition, analysis, and quantification of STAT1 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. Stained HeLa cells before and after STAT1 activation. Top panels: STAT1 localization in unstimulated cells. Bottom panels: STAT1 localization in cells stimulated by IFN-γ (5 ng/ml for 30 minutes).

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