Fluorescence images for quantitative analysis of the number of cells and morphological changes changes With Ver Changes in neuronal differentiation in the associated development time. Minimize cell clumping and SP600125 the intersection of neurites in 96 or 384-well plates for a period of up to 4 days, the cells were sparse cell/cm2 4000 seeded t.Imaging with an automated microscope with objective ImageXpress 5000A 4x equipped erm Glichte acquisition s Mtlicher into the wells of a 384-well plate in an image with sufficient resolution and high to accurately detect and quantify the various properties of the K Body neurites and cell. A typical map pixel segmentation masks generated by the software MetaXpress in Figure 2A.
Zellk were Body as Bl skirts of pixels with less than 200 m2 and identifies the maximum width of 40 m, and neurites were then identified as objects of the line more than 10 meters and at every point battery. The average length L Of neurites per cell for each well was used as a single parameter that quantifies represented as average Zellengr E outgrowth. We made variation in cell density due to the sowing or the antiproliferative effect of compounds represented by quantifying neurite outgrowth on a per cell basis. The use of automated image analysis is an accurate phone start-up Tzung the morphological properties of hundreds of cells per well allowed, without prejudice to the human cells to measure the properties.
We investigated the robustness of the automatic detection of neurites by comparing the dose-response of PC12 cells in ErbB4 NRG1 GFP and NGF as a function of time with the image recorded every 24 h, both NGF stimulates and NRG1 a continuous increase in the average Neuritenl Length on a course of four days time. NGF-treated cells appeared to distinguish slower than NRG1-treated cells within 24 hours but after four days the average L Of neurites per cell length in both treatments Similar. This sp-run response to NGF, but anything similar overall effect after four days, k Nnte secondary through upregulation of the expression of tyrosine kinase receptor TrkA or NGF receptor Ren explained To be heard, like p75NTR, which is known to stimulate the activity t to potentiate of TrkA by the formation of a receptor with high affinity t of NGF. The average length Neuritenl Strongly with the dose of NRG1 or NGF added correlated to cells particularly in concentrations of 10 ng / ml within two days and less than 20 ng / ml within four days.
These data suggest that we use automated microscopy to the L Neurite length as a Ph Genotype screening having established methods for the quantitative measurement of induced measure PC12 neuritogenesis NRG1 ErbB4 GFP cells before they are on a screen for chemical modulators We sought to understand better the signaling events with NRG1 ErbB4 signaling in order to help characterize potential downstream of all connected probes, which can be identified k. To activate ErbB4 homodimers mediate NRG1 the formation of dimers consisting ErbB ErbB3 and EGFR. W So while for ErbB4 NRG1 is required to neurite outgrowth rdern f, As all members of the ErbB family in PC12 cells expressed GFP ErbB4 remained unclear whether the other ErbB receptors also played a R The. To specifically dependent on the contribution of other ErbB family members in NRG1 ErbB4 Independent neurite outgrowth, we