The NF-κB readout: p65-GFP reporter cells A549 SIB01 and automated microscopy. a) Workflow of automated microscopy and picture analysis. Cells are fixed and stained with Hoechst 33342. Pictures are then acquired with the Scan^R system and nuclear (blue) and cytoplasmic (red) areas are defined in the Scan^R software. One activated (top) and one non-activated cell (bottom) is depicted. b) Translocation assay using Scan^R Analysis. Cells were seeded on 96-well plates, activated with TNFα (10 ng/ml), fixed, stained with Hoechst 33342 and then analyzed with automated microscopy. Scatter plots as depicted in the analysis software are shown. Cells are gated for circularity and size (Region R01), intensity of GFP and standard deviation of GFP intensity (Region R02). Cells in regions R01 and R02 are classified as active or inactive according to nuclear and cytoplasmic GFP intensity (Region R03 or R04). Cells with nuclear p65-GFP are also in region R03, whereas cells with mainly cytoplasmic p65-GFP are also in gate R04.
Bartfeld et al. BMC Cell Biology 2010 11:21 doi:10.1186/1471-2121-11-21