NFkB Activation Kit

• All reagents and protocols included for optimized sample preparation
• No cell lysis, purification, or filtration steps
• Fluorescent conjugates; no handling of radioactive isotopes
• Cells prepared with the HCS Reagent Kits may be analyzed directly via standard fluorescence microscopy; or HCS imaging system

The NFkB Activation HCS Reagent Kit has been optimized for quantification of NFkB activation by directly measuring the spatial translocation of NFkB from the cell cytoplasm to the nucleus. The kit provides a fixed end-point assay based on immunofluorescence detection of NFkB in cells grown on standard high-density microplates. The core reagents supplied include a rabbit primary antibody specific for NFkB and a DyLight™ 488-Conjugated Secondary Antibody. The cell nucleus is identified by DNA-specific Hoechst Dye, also included in the kit.

Nuclear factor kappa B (NFkB) is a transcription factor that is activated in response to ligands, such as IL-1α and tumor necrosis factor, and is associated with the activation of many cellular defense genes. Normally, NFkB is present in the cytoplasm as a complex with members of the IkB inhibitor family. Both the size of this complex and IkB masking of the nuclear localization sequence on NFkB, prevents NFkB entry into the nucleus. Upon phosphorylation and degradation of IkB, the nuclear localization sequence becomes accessible and NFkB translocates to the nucleus. NFkB must be in the nucleus to induce specific gene expression; therefore, translocation from the cytoplasm to the nucleus is a definitive measure of NFkB activation.

The NFkB Activation HCS reagents, in combination with the ArrayScan HCS Reader and the Cytoplasm to Nucleus Translocation BioApplication software afford automated plate handling, focusing, cell image acquisition, analysis, and quantification of NFkB activation. For a more detailed description of the image processing algorithm, see the Cytoplasm to Nucleus Translocation BioApplication Guide that accompanies the Cytoplasm to Nucleus Translocation BioApplication software.

Figure 1. Images of immunofluorescently stained HeLa cells before and after activation of NFkBby IL-I.  Cells were stimulated with 25 ng/mL IL-L for 20 minutes and prepared according to the kit protocol. (Left) Image of the NFkB channel in unstimulated cells. (Right) Image of the NFkb channel in stimulated cells. Both images were acquired from the ArrayScan® HCS Reader.

Figure 2. Diagrammatic view of the principle of the NFkB activation. NFkB (shaded), normally present in the cytoplasm, translocates to the nucleus upon stimulation by a variety of inducers including IL-Ix.

Figure 3. Dose response curve of IL-Iα activation of NFkB translocation in HeLA cells.

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