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.
George
Thaddeus C
TC
Amnis Corporation, Seattle, Washington.
Morrissey
Philip J
PJ
Amnis Corporation, Seattle, Washington.
Cui
Chongwei
C
University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey.
Singh
Sukhwinder
S
University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey.
Bocarsly
Patricia Fitzgerald
PF
University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey.
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D012107:Research Design
D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins