Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with Tumor Cell-Conditioned Medium

Summary

This protocol describes the dissection and culture of adult mouse dorsal root ganglion (DRG) neurons and their functional and molecular assessment following treatment with plain or tumor-conditioned medium generated from malignant peripheral nerve sheath tumor (MPNST) cells.

Abstract

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. 

Introduction

Tumor-neuron interactions are increasingly recognized as important drivers of nociceptor activity, tumor progression, and peripheral sensitization^1,2,3,4. Secreted factors released by tumor cells, including cytokines, growth factors, neurotransmitters, and metabolites, can directly or indirectly modulate the physiological state of sensory neurons, and inducing injury-related pathways, contributing to the complex interaction between tumors and the peripheral nervous system^5,6. This mo....

Protocol

DRG dissection was executed in compliance with the Institutional Animal Care and Use Committee (IACUC) at the University of Texas MD Anderson Cancer Center. C57BL/6J mice aged 8 weeks were used for the study. The overall workflow for DRG dissection, culture, treatment with conditioned medium, and subsequent functional and molecular analyses is summarized in Figure 1. The reagents and the equipment used are listed in the Table of Materials.

1. Coating coverslips and preparation

1. Prepare laminin-coated coverslips
   1. Place sterile 12 mm glass coverslips into a ....

Results

Adult DRG neurons attach efficiently to laminin-coated coverslips and exhibit round cell bodies with visible neurite outgrowth within 24 h of plating, consistent with healthy neuronal cultures. Preparations in which neurons fail to adhere, display limited neurite extension, or exhibit irregular morphology represent suboptimal outcomes and are typically excluded from downstream analysis.

Calcium imaging provides a functional readout of neuronal responsiveness. In successful cultures, brief expo.......

Discussion

This protocol provides a reproducible and flexible approach for studying how tumor-derived secreted factors influence adult sensory neuron function. Several critical steps affect the quality of isolated DRG neurons, including maintaining cold conditions during dissection, optimizing Liberase digestion time, and minimizing mechanical stress during trituration. Proper laminin coating and careful plating are essential for neuronal adhesion and survival during the first 24 h¹⁰.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell strainer, 70 µm	Corning	431751
24-Well Plate	Corning	3524
Alexa Fluor 488 goat anti-mouse IgG	Invitrogen	A11001
Alexa Fluor 647 goat anti-rabbit IgG	Invitrogen	A21244
ATF3 antibody	Novus Biologicals	NBP1-85816
Beta III Tubulin antibody (TU20)	Abcam	ab7751
Coverslips, german glass, 12 mm	Electron Microscopy Sciences	72196
Culture dishes, 100 mm	Corning	430167
Culture dishes, 35 mm	Corning	430165
DMEM, High Glucose	Corning	10-017-CV
DPBS (1x)	Corning	21-031-CV
Dumont forceps, curved (#7)	Fine Science Tools	11274-20
Dumont Forceps, straight (#5)	Fine Science Tools	11252-40
Fetal bovine serum (FBS)	Gibco	26140079
Fine Scissors - Sharp	Fine Science Tools	14060-10
Fluorescence mounting medium (Fluorsave)	Millipore	345789
Fura-2, AM	Invitrogen	F1221
Laminin	Corning	354232
Liberase DH	Roche	5401054001
Normal goat serum	Jackson ImmunoResearch	005-000-121
Paraformaldehyde, 4%	Electron Microscopy Sciences	1574
Penicillin-Streptomycin	Gibco	15140122
Pluronic F-127	Thermo Fisher Scientific	P3000MP
Stereomicroscope (dissecting), SMZ1270i	Nikon	SMZ1270i
Surgical Scissors - Straight/Sharp-Blunt/13cm	Fine Science Tools	14000-13
Triton X-100	Sigma-Aldrich	X100
Trypsin-EDTA	Sigma-Aldrich	T4049
Vannas spring scissors	Fine Science Tools	15018-10