Measurement of cytoplasmic to nuclear translocation

A method is described for the quantitative assessment of the translocation of signaling molecules from the cytoplasm to the nucleus in cells. This method utilizes fluorochrome-conjugated antibodies to the signaling molecule and a nuclear dye, and it is based on imagery acquired rapidly in flow with the use of a multispectral imaging cytometer. The analysis correlates the spatial distribution of the stained translocating signaling molecule with nuclear staining, and it generates a quantitative score for each cell using Pearson's correlation coefficient. Examples described in this section use reagents that detect NFkappaB and IRF-7 and measure the translocation of these molecules under stimulating conditions. A protocol for combining cell surface phenotype with cytoplasm to nuclear translocation is also included.