An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Begin with a multi-well plate containing a permeable membrane insert coated with poly-D-lysine.

Add astrocyte growth medium to the well and seed astrocytes, a specialized glial cell, into the insert.

Incubate to allow cell adherence on poly-D-lysine via electrostatic interactions and form a monolayer.

Replace the astrocyte medium with a neuron culture medium.

Transfer this insert into a multi-well plate containing adhered primary mouse embryonic neurons over a polymer-coated coverslip.

Incubate to establish an indirect neuron-astrocyte co-culture, where the two cell types are physically separated yet share the same culture medium.

Astrocytes secrete various neurotrophic factors essential for neuron survival and growth.

These factors migrate through the permeable support and interact with primary neurons. This promotes their differentiation and forms neuronal projections.

These projections elongate and establish synaptic connections between neurons, indicating neuron-glial interactions.