Journal: bioRxiv

Article Title: Macrophage-specific NF-κB activation dynamics can segregate inflammatory bowel disease patients

doi: 10.1101/535096

Figure Lengend Snippet: Time-lapse Confocal imaging of NF-κB nuclear translocation in Lipid A-stimulated human PBMDMs. (A) PBMC-derived macrophages from human subjects were transduced with a fluorescent lentiviral p65AmCyan construct and were used to image NF-κB p65 subunit cellular dynamics in real-time. (B) CellTracker was used to generate profiles of nuclear translocation of transduced cells post stimulation with 200ng/mL Lipid A, over 3h: Asterisks (*) indicate nuclear localization of p65 at 66 and 93 min (‘). (C-E) Graphs of individual nuclear translocation profiles of responding cells in the healthy controls, Crohn’s disease (CD) and ulcerative colitis (UC) groups; mean ± SD for all responsive cells shown in blue, red and green colour lines for each group, respectively. (F) The proportion of responding cells was calculated by pooling all tracked cells from each disease group; *p<0.05; two-sided Fisher’s exact test (by summation). Peak identification and analysis revealed in each population the peak time (G), peak amplitude (H) and peak width (I): *p<0.05, ***p<0.001; statistical differences assessed by Kruskal Wallis test with pairwise comparison of all three groups.

Article Snippet: Cells were imaged using a Zeiss LSM880 confocal microscope system equipped with a cell incubation unit maintained at 37 ° C, in a humidified atmosphere of 5% CO 2 . p65-AmCyan nuclear fluorescence was detected (excitation λ 458nm, emission λ 489nm) and quantified using CellTracker software ( ).

Techniques: Imaging, Translocation Assay, Derivative Assay, Transduction, Construct, Comparison