Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.
Nuclear translocation of NF-kappaB 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-kappaB images from each cell. This approach accurately measures NF-kappaB translocation in cells with small cytoplasmic areas in dose- and time-dependent manners. Further, NF-kappaB 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.
George
Thaddeus C
TC
Amnis Corporation, 2505 Third Ave., Suite 210, Seattle, WA 98121, USA. t.george@amnis.com
Fanning
Stacey L
SL
Fitzgerald-Bocarsly
Patricia
P
Fitzgeral-Bocarsly
Patricia
P
Medeiros
Ricardo B
RB
Highfill
Sarah
S
Shimizu
Yoji
Y
Hall
Brian E
BE
Frost
Keith
K
Basiji
David
D
Ortyn
William E
WE
Morrissey
Philip J
PJ
Lynch
David H
DH
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000276:immunology*; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D003593:Cytoplasm / Q000276:immunology; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D050839:Interferon Regulatory Factor-7 / Q000276:immunology; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000276:immunology* / Q000378:metabolism
D050839:Interferon Regulatory Factor-7; D008070:Lipopolysaccharides; D016328:NF-kappa B; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D016650:Fluorescein-5-isothiocyanate