Nevertheless, recent innovations in analysis of genome-wide interaction networks or functional ��interactomes��, have placed unprecedented emphasis on understanding how XAV939 chromatin dynamics facilitate the formation of gene interactions networks, which in turn might contribute to the regulation of gene expression in mammalian cells . If long-range chromosomal interactions make a significant contribution to the regulation of gene expression in higher eukaryotes, it is important to understand the range and extent of interactions that this involves. To address this issue, we have used single cell imaging techniques to monitor chromatin mixing in human HeLa cells. DNA foci were pulse-labeled using fluorescent dNTP analogues that incorporate during replication and remain stably associated with labeled CTs for at least 14 days. After labeling, mitotic segregation reveals discrete chromatin domains with clearly defined DNA foci, so that the dynamic properties of foci and interactions between foci of neighboring CTs can be assessed. We show that while Wortmannin individual foci are spatially dynamic their DNA is generally locally constrained and so limits mixing between neighboring CTs. We tested the extent of DNA mixing between CTs using established protocols that allow CTs and individual DNA foci to be visualized in living cells . Cells were pulse-labeled with AF488-dUTP, grown for 24 h and then pulse-labeled with Cy3- dUTP and grown for a further 1�C2 days . Because replication is semi-conservative and mitotic chromosome segregation is random, this protocol yields cells that contain uniquely red or green labeled CTs together with a minority of CTs that are unlabeled . Live cell analysis showed that the identity of CTs is preserved for many hours with little or no interaction between neighboring CTs. However, as resolution is limited by the low levels of illumination used during live cell imaging we also performed imaging on fixed cells . Post-fixation analysis, in the absence of processing that might perturb chromosome structure at the resolution analyzed by light microscopy, allows the structure of the differentially labeled chromatin domains and distribution of their foci to be visualized . With this type of analysis, the structure of DNA foci is clearly preserved and foci are clustered into local domains that represent individual or small groups of CTs. Notably, the boundaries between adjacent green and red domains are clearly defined and regions of apparent co-localization between the differentially labeled regions were restricted to these boundary domains. However, rotation of the 3D image suggested that many sites of apparent localization resulted from the spatial overlap of adjacent foci in projections of optical sections and not true co-localization within individual voxels of the 3D image .