Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages.
Quantitative measurement of NF-kappaB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-kappaB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-kappaB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-kappaB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-kappaB activation reporter cell line.
Department of Immunology & Molecular Pathology, University College London, Windeyer Building, 46 Cleveland Street, W1T 4JF London, UK. m.noursadeghi@ucl.ac.uk
Mahdad
M
Noursadeghi
Jhen
J
Tsang
Thomas
T
Haustein
Robert F
RF
Miller
Benjamin M
BM
Chain
David R
DR
Katz
D021581:Active Transport, Cell Nucleus; D018929:Cell Culture Techniques; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D055666:Lipopeptides; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D015203:Reproducibility of Results; D012815:Signal Processing, Computer-Assisted; D012984:Software; D013053:Spectrophotometry; D013997:Time Factors; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D055666:Lipopeptides; D008070:Lipopolysaccharides; C508075:Pam(3)CSK(4) peptide; D010455:Peptides; D051195:Toll-Like Receptor 2; D051996:Transcription Factor RelA; D011112:Polymyxin B