HCS image with assay features shown as an overlayOptimized, automated assays for HCS

Nuclear factor kappa B (NFκB) is an important transcription factor in physiological processes such as cell proliferation and cellular responses to stress. Normally, NFκB is present in the cytoplasm, and translocation from the cytoplasm to the nucleus is a definitive measure of NFκB activation. In this automated assay, translocation of the activated transcription factor from the cytoplasm to the nucleus is automatically detected and quantified for each cell. The assay is conveniently run using three detection channels, with a nuclear stain, a whole-cell stain, and a specific label for the transcription factor.

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Typical transcription factor activation assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield
  • 10x or 20x magnification

Automatically measured properties

  • Transcription factor intensity in the cytoplasm and in the nucleus
  • Difference in transcription factor intensity between the cytoplasm and nucleus, as a measure of cytoplasm-to-nucleus translocation
  • Ratio of transcription factor intensity between the cytoplasm and nucleus, as a measure of cytoplasm-to-nucleus translocation
  • Percentage of cells in the well above a user-definable threshold for the nucleus-to-cytoplasm intensity difference or ratio for one or more targets
  • Nuclear shape and dimensions

Transcription factor activation assay images

Transcription factor activation (Left) HeLa cells were treated with TNFα, fixed, permeabilized, and stained with Invitrogen HCS NuclearMask Blue (blue), and the NFκB was labeled by indirect immunofluorescence (green). (Right) Cell features for the assay were automatically detected using Thermo Scientific HCS Studio Software: nucleus (blue overlay), cytoplasm (green overlay), and NFκB intensity. The red overlay shows the cells where translocation did not occur.

NFκB cytoplasm-nucleus intensity difference vs. ratio. NFκB intensity differences between the cytoplasm and nucleus were automatically calculated and plotted. Red dots in the scatter plot of the NFκB cytoplasm-nucleus intensity difference versus ratio correspond to cells that did not exhibit NFκB translocation.


Sample experimental data


Dose-response plot showing the activation of NFkB by increasing TNFα concentration.
The y-axis shows the intensity difference between the nucleus (containing translocated NFkB) and the surrounding cytoplasm (containing un-translocated NFkB) as a measure of NFkB translocation.

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