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Quantitating NFkB translocation using the Amnis FlowSight imaging cytometer and optimized reagent kit


Merck:/Freestyle/BI-Bioscience/Cell-Analysis/amnis/Cell-signaling-100x100.jpgMolecular translocation of transcription factors from the cytoplasm to the nucleus is a pivotal event in many processes critical to cellular activation, differentiation, and host defense. Existing methods used to measure nuclear translocation have significant limitations. Amnis® imaging flow cytometry overcomes these limitations by automatic, rapid collection of images that can be objectively quantified in solution. The ability to numerically score large numbers of automatically acquired images is ideally suited to the analysis of nuclear translocation within many cell types including small, primary immune system cells.

Featured Video

Watch to learn how multispectral imaging in flow is being used to answer specific cell signaling research questions. Dr. Sherree Friend explains how Amnis® applications use high-throughput imaging of cell signaling events such as translocation of NFκB during inflammation. The advantages of imaging flow cytometry are compared with signaling assays that use Western blot and microscopy.

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HIV-Specific Translocation of NFAT

HIV-specific nuclear localization of NFAT was measured in HIV-experienced T cells from peripheral blood of HIV+ patients. HIVgag peptide specifically stimulates NFAT (green) to move to the nucleus (red) in HIV-tetramer positive cells (orange). The Similarity score correlates DRAQ5 nuclear stain with the NFAT signal. The higher the Similarity score, the more translocation is visualized in the example images from each quadrant.

NF-κB Translocation in T:APC Conjugates

Measurement of NFκB translocation in transgenic T cells which are in contact with antigen presenting cells (APC) and specific peptide. Specific T cell:APC conjugates are identified and translocation of NFκB from the cytoplasm to the nucleus specifically within the T cells is measured in the presence (shown) or absence of peptide.

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