Comprehensive protocol for culturing and functionally characterizing primary mixed neural cells from the neonatal rat cortex.

In vitro models using purified neurons or glial cells are crucial for studying neurological functions but often overlook intercellular interactions. Mixed neural cell cultures offer a more physiologically relevant system by preserving cell-to-cell communication and providing deeper insights into neural behavior. Here, we present a protocol for culturing mixed primary cells from the neonatal rat cerebral cortex and functionally characterizing them via calcium imaging. This method enables cell phenotyping, spatial distribution analysis, and activity monitoring in response to stimuli. Our model maintained a cellular composition resembling the native rat cortex, with 35.4 % neurons, 44.3 % astrocytes, and 20.3 % other cell types. Calcium imaging showed that ATP (100 μM) and BzATP (100 μM) evoked stronger calcium transients than KCl (50 mM). BzATP induced a sustained response mediated by P2X7 receptor activation, while ATP activated a broader range of P2 receptors. Unlike purified or enriched cultures, this mixed-cell system better replicates the cellular environment of the brain, ensuring reproducibility and biological relevance. This protocol provides a straightforward platform for investigating neuron-glia interactions and neural signaling, bridging the gap between simplified in vitro models and the complexity of neural networks. Its applications may advance research into neurobiological disease mechanisms and therapeutic development.