Multiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers

Abstract

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.Key features• Preparation and attachment of PDMS microfluidic chambers to glass coverslips.• Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber.• Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb).• Immunofluorescence and multilabeling of compartmentalized cortical neurons.• Retrograde transport of fluorescently labeled BDNF.