A method to assess multiple aspects of the motile behaviour of adherent PC12 cells on applied biological substrates.

摘要

Cellular migration is central to a wide range of biological and pathological processes in vivo. In vitro cell migration assays can be used to obtain invaluable information relating to the mechanism of cell movement, but current available methods can be limiting. Here we describe a novel motility assay that allows the simultaneous investigation of both quantitative and qualitative aspects of a population of motile cells as they move across a variety of substrates. By plating cells in a confluent monolayer on a coverslip, the monolayer can then be inverted to migrate over a larger substrate-coated coverslip, which can subsequently be reliably quantified, and subjected to immunocytochemistry and confocal imaging. This assay can be used to assess multiple aspects of motility, including distance, quantity, morphology, polarization and component colocalization. To demonstrate the utility of this assay, it was applied to the study of a stimulator of PC12 cell migration, nerve growth factor (NGF), and how thismigration is influenced by the extracellular substrate, laminin. Furthermore, since mutations to the NGF receptor, TrkA, have been noted to alter the behaviour of PC12 cells in response to NGF, a PC12 subline that expresses a mutated TrkA receptor was utilized to illustrate that a Y785F mutation in the cytoplasmic tail of TrkA results in increased migration in response to the stimulus compared to the control PC12s.