The fluorescent reagent, NeuroLight Red™, specifically labels both primary neurons and human iPSC derived neurons when grown in co-culture with astrocytes, allowing for quantitation using the IncuCyte NeuroTrack™ software. In this tutorial, we describe a method for visualizing and quantitating neurite outgrowth in co-culture with astrocytes, utilizing a novel fluorescent label for neuronal processes and the IncuCyte ZOOM live-cell imaging system. However, a limitation of a monoculture approach is that it does not detect the effects of glial cells on neurite development and function. This can be achieved in neuronal monocultures using phase images obtained with Essen Bioscience’s IncuCyte ZOOM™ live-cell imaging platform followed by analysis with IncuCyte NeuroTrack™ software. An ideal in vitro method to track neurite dynamics would allow continuous, automated measurement of structural parameters, including length and number of branch points, in a nonperturbing manner.
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