Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with...

Interactions between sensory neurons and tumor cells are increasingly recognized as critical contributors to tumor progression and cancer-associated pain. Here, we describe a reproducible protocol for the isolation and culture of adult mouse dorsal root ganglion (DRG) neurons, followed by functional and molecular assays to assess their responses to tumor-derived secreted factors. Adult DRG were dissected, enzymatically digested with Liberase DH, and dissociated into single neurons. The cells were plated on laminin-coated coverslips and maintained in defined culture conditions that promote neuronal survival while minimizing glial overgrowth. Subsequently, the cultured neurons were exposed to tumor cells' conditioned medium to model paracrine signaling at the neuron-tumor interface. Functional responses were monitored by live cell calcium imaging using fluorescent indicators, enabling quantification of neuronal activity and stimulus-specific responsiveness. In parallel, neuronal stress and injury are evaluated by ATF3 immunostaining. Together, these complementary approaches provide a powerful platform to investigate how tumor-secreted factors sensitize or activate sensory neurons. This protocol integrates adult DRG culture, tumor-conditioned medium treatment, calcium imaging, and molecular readouts into a single workflow, enabling researchers to dissect mechanisms of neuron-tumor communication. The method is broadly applicable to studies of cancer neuroscience, pain signaling, and the identification of secreted mediators that modulate neuronal function in disease contexts.