Nuclear translocation of NF-κB initiates transcription of numerous genes, many of which are critical to host defense. Fluorescent image-based methods that quantify this event have historically utilized adherent cells with large cytoplasm-to-nuclear area ratios. However, many immunologically relevant cells are naturally non-adherent and have small cytoplasm-to-nuclear area ratios. Using the ImageStream® imaging flow cytometer, we have developed a novel method that measures nuclear translocation in large populations using cross-correlation analysis of nuclear and NF-κB images from each cell. This approach accurately measures NF-κB translocation in cells with small cytoplasmic areas in dose- and time-dependent manners. Further, NF-κB translocation was accurately measured in a subset of cells contained in a mixed population and the technique was successfully employed to measure IRF-7 translocation in plasmacytoid dendritic cells (PDC) obtained from human peripheral blood. The techniques described here provide an objective and statistically robust method for measuring cytoplasmic to nuclear molecular translocation events in a variety of immunologically relevant cell types with characteristically low cytoplasm-to-nuclear area ratios.
Bibliographical noteFunding Information:
Development of the ImageStream® technology was partially supported by NIH grants 9 R44 CA0l798-02 and 1 R43 GM58956-01.
- Image cytometry
- Signal transduction