1 Introduction Ghosh et al., 1998 32 6 Deptala et al., 1998; George et al., 2006; Rogers and Fuseler, 2007; Fuseler et al., 2006 3 2 Methods 2.1 Macrophage culture and innate immune stimulation l 7 5 3 2.2 Immunofluorescence staining 2 2.3 Image acquisition and analysis Fig. 1 http://rsb.info.nih.gov/ij Fig. 1 Ridler and Calvard, 1978 Fig. 1 Fig. 2 Fig. 2 3 Data presentation and discussion Fig. 3 Fig. 4 3 Fig. 5 3 . Fig. 6 r 2 The method proposed here is most suitable for adherent cell cultures with relatively large cytoplasmic:nuclear area ratios that allow clear distinction between nuclear and cytoplasmic NF-κB staining. It requires relatively few cells and can be used to study NF-κB nuclear translocation at single-cell level or in mixed cultures. It can be readily applied to the study of NF-κB activation in macrophages, dendritic cells, epithelial and endothelial cells, and fibroblastic cells. We have also been able to apply this method to monocytic cells in suspension (THP-1 and K562 cell lines) by air drying them onto coverslips for immunostaining (data not shown), albeit their typically small nuclear:cytoplasmic area ratio may limit the accuracy of quantitation. This generic image analysis methodology may be applied to quantitative analysis of other transcription factors and signalling events in which assessment of sub-cellular localisation is necessary. Where confocal microscopy facilities are available, this method overcomes the problems related to sensitivity, use of radioisotopes and cost. It can be easily adopted in current cellular immunology research, and given the ready accessibility of the public domain image analysis software, with further validation this methodology may serve as a universal standard that allows better comparison of data from separate experiments and different research groups.