Measurement of TLR-Induced Macrophage Spreading by Automated Image Analysis: Differential Role of Myd88 and MAPK in Early and Late Responses.

摘要: Sensing of infectious danger by Toll-like receptors (TLR) on macrophages causes not only a reprogramming the transcriptome but also changes in cytoskeleton important for cell spreading and motility. Since manual determination contact areas from fluorescence microscopy pictures is very time consuming prone to bias, we have developed tested algorithms automated measurement macrophage spreading. The two-step method combines identification cells nuclear staining with DAPI surface integrin CD11b. Automated image analysis correlated well annotation resting early after stimulation, whereas at later points segmentation algorithm showed slightly larger variation. was applied investigate impact genetic or pharmacological inhibition known TLR signaling components. Deificiency adapter protein Myd88 strongly reduced activity late points, had no LPS stimulation. A similar effect observed upon MEK1, kinase activating MAPK ERK1/2, indicating that ERK1/2 mediates Myd88-dependent In contrast, lacking p38 were impaired initial response responded normally 8 – 24 h dichotomy effects raises question which respective substrate proteins mediate(s) cytoskeletal remodeling described here increases objectivity greatly reduces required such investigations therefore expected facilitate through-put spreading, e.g. siRNA knockdown screens.