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Molecular Translocation BioApplication |
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Catalog No.S50-0019-2 DescriptionBiological signal transduction is a complex process that involves activation and translocation of multiple signaling molecules. The majority of signaling events involve multiple interacting components and, in many instances, activation is coupled to movement of a target molecule from one location of a cell to another, transmitting a biological signal in the process. Building on the success of the Cytoplasm to Nucleus Translocation BioApplication, we have added more measurement channels, enhanced flexibility, and more focused output features to create the Molecular Translocation BioApplication. It is capable of measuring translocation events for up to 5 different fluorescently labeled targets with an additional channel used for object identification and definition of the cellular region of interest (Figure 1). It can simultaneously quantify the activation and translocation of a combination of multiple transcription factors and/or kinases such as NFkB, NFAT, c-jun, p38, and ERK, and analyze the interactions between related or interacting signaling pathways – all in the same cell. This capability is a key requirement for pathway profiling. and dissection of complex signaling events. The Thermo Scientific Cellomics Molecular Translocation BioApplication provides the following enhancements relative to traditional methods for analysis of transcription factor activation: - Increased throughput compared to gel-shift assays
- Automation compatible method
- Does not require generation of specific reporter cell lines
- Does not require isotopes or cell lysis
- Visualization of activity in single cells
- Ability to define specific cellular sub-populations for analysis
Using sophisticated image processing methods, the Thermo Scientific Cellomics high content screening platform offers the unique ability to quantify intracellular movement of fluorescently labeled target molecules within single cells in a fully automated fashion. The Molecular Translocation BioApplication delivers a versatile solution that allows the user to flexibly define a cellular or sub-cellular region of interest with a fluorescent marker, and then monitor translocation events to that domain for up to five separate target molecules. Users are able to define activation thresholds for multiple targets and the percentage of cells exceeding thresholds are automatically reported by the application. The Molecular Translocation BioApplication was utilized in the following example for simultaneous measurement of NFkB and c-jun activation in HeLa cells. Combining the analysis of two or more targets such as these into a single assay allows accurate profiling of signaling pathways at the single cell level. Cells were incubated with varying concentrations of either anisomycin or TNFa and immunolabeled microplates were scanned using the ArrayScan HCS Reader. This multiparameter experiment allows one to identify compounds capable of activating both target proteins. The data presented in Figure 2 shows the well-level results for each individual transcription factor, as seen in the Thermo Scientific Cellomics vHCS:View visualization software.
The dose-response curves from individual rows are overlaid in this example to indicate the reproducibility of the measurements. The simultaneous analysis of both phospho-c-jun and NFkB following stimulation with either TNFa or anisomycin is presented below in Figure 3. The data demonstrate that anisomycin causes translocation of both transcription factors in only a small percentage of cells, whereas treatment with TNFa induces translocation of NFkB and c-jun in a much higher percentage of the cell population. This BioApplication offers a very powerful approach for analysis of transcription factor and kinase activation by monitoring movement between the cell cytoplasm and the nucleus.
Features- Specifically identifies neuronal cells in a mixed culture and provides objective analysis of neurite outgrowth changes.
- Multi-parametric analysis of up to five target molecules
- Reports differences and ratios of fluorescence intensity between subcellular regions of interest
- Reference wells can be used to set thresholds for nuclear-cytoplasmic difference and ratio measurements
- Automatically computes % of cells in each well that are above upper threshold or below lower threshold for main output features
- Intensity gating capability enables isolated analysis of a sub-population of cells
Benefits- Unique assay for analysis of translocation events not possible with other systems
- Greater data quantity and quality from a single assay plate
- Streamlined for focused study of multiple cellular translocation events
- Correlates multiple translocation events in individual cells
- Sophisticated threshold selection allows users to correlate % of responders across multiple targets
- Isolates and analyzes specific cellular sub-populations within mixed cultures
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