NFAT Activation Kit

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 NFAT Activation Kit
 Product# Price  Product Name  Pack Size  
         
 K0100111      -    NFAT-1 (green) Activation  5 x 96    SELECT   
 R0105151      -    NFAT-1 (green) Activation  50 x 96    SELECT   
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Figure 3. Dose-responsiveness of anisomycin (in ng/ml) vs average cytoplasmic to nuclear translocation differences against seven different cell types.

Figure 2. Diagrammatic view of the principle of the ATF-2 Activation reagent kit. NFAT (green), normally present in the cytoplasm, translocates to the nucleus upon dephosphorylation via binding to calcineurin.

Figure 1. Images of HeLa cells before (left) and after (right) activation of NFAT by thapsigargin. Cells were stimulated with 60nM thapsigargin for 40 min, and then prepared using the kit reagents and protocol.  Nuclei appear blue (Hoechst) and NFAT appears green. Images were aquired by the ArrayScan? HCS Reader.

The NFAT-1 Activation Kit protocol was optimized for a fixed end-point assay. Inhibitors of NFAT-1 translocation are screened by stimulating cells with a control inducer, such as thapsigargin, after exposing live cells to the test compounds. Replacing thapsigargin in the assay with test compounds identifies agonists of NFAT-1 translocation. Translocation is directly quantified as the difference in cytoplasmic to nuclear intensity of the labeled transcription factor. The NFAT-1 Activation Kit, in combination with the Thermo Scientific ArrayScan? HCS System and the Cytoplasm to Nucleus Translocation Application software affords automated plate handling, focusing, cell image acquisition, analysis, and quantification. For more detailed description of the image processing algorithm, see the Cytoplasm to Nucleus Translocation Application Guide that accompanies the software.

Transcription factors belonging to the NFAT family regulate cytokine expression in T cells and are intimately involved in regulation of early events of the immune response. NFAT resides in the cytoplasm and translocates to the nucleus upon dephosphorylation via binding to calcineurin (calcium/calmodulin-dependent phosphatase). Upon translocation, NFAT forms complexes with AP-1 proteins to regulate gene expression.

Cell-based assay provides an earlier and more definitive measure of NFAT activation relative to electophoretic mobility shift or gene reporter assays
Immunofluorescence-based screening assay provides sensitive quantitative and qualitative results without radioactivity
Protocol requires no cell lysis, purification, or filtration steps
Cells prepared with the HCS Reagent Kits may be analyzed directly via standard fluorescent microscopy; or HCS imaging systems.
The Cellomics NFAT-1 Activation Kit provides the reagents necessary to quantify NFAT-1 activation by directly measuring its translocation from the cytoplasm to the nucleus. The assay is performed on live cells growing on standard high-density microplates. The kit is supplied with an anti-NFAT-1 primary antibody and a DyLight 488-conjugated Secondary Antibody. The nuclear region is identified by Hoechst Dye, also included in the kit.

 
NFAT Activation Kit Features

   Product Detail 

The NFAT (nuclear factor of activated T cells) Activation HCS Reagent Kit provides for high-content screening (HCS)-quality fluorescence reagents along with a validated, optimzied protocol for measuring and visualizing the activation of the NFAT transcription factor.