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 modulation plays a significant role in cancer-associated neuropathic pain and can influence tumor progression and immune responses⁴. Reciprocally, neuronal activity has been shown to influence tumor growth and metastatic behavior⁷. Understanding the molecular and functional consequences of exposure to tumor-derived signals requires experimental systems that enable controlled manipulation of the neuronal microenvironment.

Dorsal root ganglion (DRG) neurons are the primary sensory neurons responsible for transducing nociceptive, mechanosensory, and thermosensory information. Adult DRG neurons maintain subtype-specific molecular identity, receptor expression, and electrophysiological characteristics that more accurately reflect in vivo biology compared with neonatal DRG cultures⁸. Several models have been developed to study direct physical interactions between tumor cells and sensory nerves, including co-culture systems and neural invasion assays⁹.

The protocol presented here provides a robust in vitro platform for evaluating how tumor-derived secreted factors alter sensory neuron function. We describe the dissection and culture of primary DRG neurons from adult mice, the preparation of tumor-conditioned medium from mouse MPNST cells, and the use of live-cell calcium imaging to assess functional responses. Complementary immunostaining for ATF3 provides a molecular readout of injury and activation signaling within DRG neurons. This approach allows investigators to dissect mechanistic pathways involved in tumor-induced neuronal sensitization and to identify candidate mediators of neuron-tumor communication.

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 24-well plate or a 35 mm culture dish.
   2. Dilute laminin to a final concentration of 10 µg/mL using sterile 1x DPBS.
   3. Add 80 µL of laminin solution to fully coat each coverslip.
   4. Incubate for 45 min at room temperature in a laminar flow hood or at 37 °C in an incubator.
   5. Aspirate the remaining laminin from the coverslips immediately before seeding the DRG cell suspension.
2. Prepare Liberase digestion solution
   1. Thaw a (100 µL, 1.8 U) aliquot of Liberase DH on ice.
   2. Dilute into 900 µL of ice-cold DPBS.
   3. Keep on ice until needed.
3. Prepare buffers and medium
   1. Chill 50–100 mL of DPBS for spine transport and ganglia extraction.
   2. Warm DRG culture medium (DMEM + 10% FBS + 1x Penicillin-streptomycin) to 37 °C.
   3. Keep 15 mL of medium on ice for ganglia collection.

2. DRG dissection

1. Euthanasia and spine removal
   1. Euthanize the mouse using CO₂ or a 5% isoflurane overdose, followed by decapitation.
   2. Spray the dorsal surface with 70% ethanol.
   3. Excise the spinal column and immediately place it in ice-cold DPBS.
2. Exposure of dorsal root ganglia
   1. Divide the spinal column into thoracic and lumbar sections.
   2. Bisect each section longitudinally along the spinal canal to expose nerve trunks and connective tissue. Transfer both halves into a fresh 100 mm Petri dish with ice-cold DPBS.
3. Dorsal Root Ganglia (DRGs) Extraction (Under the dissection microscope)
   1. Cut the roots and remove the spinal cord and meninges to access the DRGs.
   2. Identify DRGs along the intervertebral foramina.
   3. Carefully excise each DRG while minimizing the inclusion of nerve roots and meninges using spring scissors and #55 fine forceps. With practice, it is typically possible to recover ~30–45 DRGs per mouse (corresponding to ~15–23 DRG pairs), depending on dissection and handling.
   4. Transfer harvested ganglia into 15 mL of ice-cold culture medium in a 100 mm Petri dish.

3. DRGs dissociation

1. Digest DRGs
   1. Transfer DRGs into 1 mL of Liberase DH solution.
   2. Incubate for 30 min at 37 °C in CO₂ incubator or a water or bead bath.
   3. Transfer digested DRGs into a 15 mL conical tube.
   4. Triturate gently using standard P1000 pipette tips for 10–15 passes until the suspension appears turbid. Begin with a larger effective bore and progressively reduce the bore size during trituration to achieve a single-cell suspension while minimizing mechanical stress. Pre-wet pipette tips with culture medium containing 10% FBS prior to use to reduce cell adhesion. Fire-polished glass Pasteur pipettes may also be used as an alternative.
   5. Stop digestion by adding 10 mL of warm culture medium.
   6. Filter the suspension through a 70–100 µm cell strainer into a 50 mL conical tube and adjust the final volume to 20 mL with fresh culture medium.
2. Pellet cells
   1. Centrifuge at 250 × g for 5 min at room temperature.
   2. Discard the supernatant.
   3. Resuspend the pellet in 500–600 µL of warm culture medium.

4. Seeding DRG cells

1. Aspirate laminin from coverslips.
2. Add 50–80 µL of neuronal suspension to each coverslip center.
   NOTE: With typical recovery of ~30–45 DRGs per mouse, enzymatic and mechanical dissociation yields sufficient cells to seed approximately 10–12 coverslips, with an average neuronal density of ~150 neurons per coverslip under the described plating conditions.
3. Incubate for 2 h at 37 °C to allow adherence.
4. Add carefully 1–2 mL of warm culture medium to each well/dish and incubate overnight.
5. Perform functional assays within 24–48 h of plating; however, recordings within 24 h are recommended to minimize glial proliferation and preserve baseline neuronal responsiveness.

5. JW23.3 MPNST cell culture and conditioned medium (CM) preparation

1. Maintain JW23.3 cells
   1. Culture the cells in complete culture medium (DMEM + 10% FBS + 1x Penicillin-streptomycin) in a 100 mm Petri dish.
   2. Incubate at 37 °C, 5% CO₂ incubator.
2. Subculture cells
   1. Wash with DPBS.
   2. Add 0.25% Trypsin-EDTA and incubate 3 min.
   3. Neutralize with complete culture medium.
   4. Collect the cells and centrifuge at 250–300 × g for 5 min at room temperature, and reseed at 2 × 10⁶ cells/mL in a 100 mm Petri dish.
3. Prepare conditioned medium (CM)
   1. Next day, wash the cells with DPBS and add 8 mL fresh warm culture medium to the cells and the plain (no cells) Petri dish. Conditioned medium is collected at a consistent confluency (~70%–80 %) using cells at similar passage numbers to minimize variability in secreted factor levels.
   2. Incubate at 37 °C, 5% CO₂ for 48 h.
   3. Collect CM and spin at 250–300 × g for 2–3 min. Simultaneously treat DRG neurons with the CM or immediately store at -80°C.

6. Treatment of DRG

1. Replace neuronal medium with CM.
2. Incubate with DRG neurons for 1–4 h, depending on assay.

7. Live-cell calcium imaging

1. Perform calcium imaging
   1. Prepare individual coverslips containing DRG neurons for dye loading.
   2. Incubate coverslips with 2 µM Fura-2 AM prepared in Pluronic F-127 (1:1) at room temperature for 30 min in the dark.
2. Mount coverslips for imaging
   1. Place each coverslip into the recording chamber and mount it on the stage of an inverted fluorescence microscope.
3. Superfuse cells with physiological buffer
   1. Continuously perfuse cells using a gravity-driven perfusion system at 1–2 mL/min with HEPES-buffered solution containing (in mM): 140 NaCl, 5 KCl, 1.3 CaCl₂, 0.4 MgSO₄, 0.5 MgCl₂, 0.4 KH₂PO₄, 0.6 NaHPO₄, 3 NaHCO₃, 10 glucose, and 10 HEPES.
   2. Adjust the buffer to pH 7.4 with NaOH and to ~310 mOsm with sucrose.
4. Set up fluorescence excitation and acquisition
   1. Alternately excite Fura-2-loaded cells at 340 nm and 380 nm using a xenon light source and controller with a 2 nm band-pass filter, 100 ms exposure time, and acquire images at 1 Hz.
5. Capture fluorescence emission
   1. Collect emission at 510 nm using a 10× objective (NA 0.5) and an sCMOS pco.edge camera.
   2. Calculate the F340/F380 ratio for each neuron using imaging software by defining regions of interest (ROIs).
6. Record baseline activity
   1. Acquire a 60 s baseline recording while perfusing cells with physiological buffer.
7. Apply CM
   1. Apply control CM or MPNST cell CM acutely during imaging for 120 s, followed by 120 s of recovery in physiological buffer.
   2. Maintain continuous perfusion during acquisition to ensure stable baseline conditions.
      ​NOTE: Conditioned medium is applied acutely during imaging, and matched control CM is used to control for non-specific effects of media exchange.
8. Confirm neuronal viability
   1. Apply a terminal depolarizing stimulus of 50 mM potassium chloride (KCl) for 60 s to confirm neuronal viability.
9. Define and analyze calcium responses
   1. Draw ROIs around the individual neurons and extract F340/F380 ratio values in a spreadsheet.
   2. Define a neuron as responsive if the peak increase in the F340/F380 ratio exceeds 0.1 above baseline during agonist application/experimental stimuli.
10. Collect sufficient replicates
    1. Acquire data from four to six coverslips per experimental group.
    2. Calculate the delta response for each stimulus condition.
    3. Plot time versus ratio using appropriate analysis software.

8. Immunostaining

1. Fix DRG neurons with 4% paraformaldehyde for 20 min at room temperature.
2. Carefully wash 1× with PBS.
3. Incubate the cells with blocking buffer 5% normal goat serum (NGS) in PBST (0.5% Triton X-100 in PBS) for 1 h at room temperature.
4. Dilute the primary mouse anti- β3-tubulin antibody (1:200) and rabbit anti-ATF3 antibody (1:200) in blocking buffer.
5. Incubate overnight at 4 °C.
6. Incubate with secondary antibodies, goat anti-rabbit Alexa Fluor 647 and goat anti-mouse Alexa Fluor 488 for 2 h at room temperature.
7. Carefully remove coverslips and mount on slides using fluorescence mounting medium.

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 exposure to conditioned medium induces detectable changes in the 340/380 fluorescence ratio, followed by recovery toward baseline after washout. Subsequent depolarization with high K⁺ elicits a rapid and robust increase in intracellular calcium (Figure 2), confirming neuronal viability and adequate dye loading. Neurons exposed to tumor cell-conditioned medium exhibit calcium responses. Suboptimal recordings are characterized by unstable baselines, low signal-to-noise ratios, or reduced responses to high K⁺ stimulation.

Immunostaining demonstrates nuclear ATF3 immunoreactivity following incubation with the tumor cell-conditioned medium (Figure 3E–H). Co-immunostaining with β3-tubulin is used to identify sensory neurons and confirm neuronal-specific ATF3 expression. Together, these representative outcomes demonstrate successful execution of the method and illustrate the range of functional and molecular responses that can be observed in cultured adult mouse sensory neurons.

Figure 1: Experimental workflow. Schematic representation illustrating DRG dissection and culture, preparation and application of conditioned medium, and downstream calcium imaging and immunocytochemistry. (A) Dissection of the mouse spine, isolation of DRGs, and enzymatic digestion followed by seeding to establish primary DRG neuron cultures. (B) Preparation of CM from MPNST cells and plain medium, followed by incubation of DRG neurons with CM. (C) Assessment of neuronal responses using calcium imaging and immunocytochemistry. Please click here to view a larger version of this figure.

Figure 2: Representative calcium imaging traces. Traces show (Mean ± SEM) changes in intracellular calcium flux (340/380 ratio) in DRG neurons treated with plain CM (blue) or MPNST CM (red), followed by washout and high K⁺ stimulation. Please click here to view a larger version of this figure.

Figure 3: ATF3 immunostaining of DRG neurons. Representative images show ATF3 (red) expression in β3-tubulin-positive DRG neurons (green) under plain CM (A–D) and MPNST CM (E–H). The white box indicates the area magnified in the corresponding inset. Scale bar, 50 µm (A–C,E–G) and 10 µm (D,H). Please click here to view a larger version of this figure.

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.

Compared with neonatal DRG cultures or immortalized neuronal cell lines, adult DRG neurons preserve mature electrophysiological and molecular characteristics that are essential for studying pain-related signaling pathways^8,10,11,12. A limitation of this approach is the absence of direct neuron-tumor contact, which may contribute additional signaling cues in vivo. However, isolating paracrine mechanisms allows investigators to identify candidate tumor-secreted mediators of neuronal activity and sensitization.

Acute application of conditioned medium enables the assessment of rapid neuronal responses to tumor-derived soluble factors, whereas pre-incubation paradigms may reflect longer-term sensitization effects. The use of matched control-conditioned medium ensures that observed responses are attributable to tumor-derived factors rather than changes in medium composition. Although neurons exposed to tumor-conditioned medium showed a trend toward reduced responses to the terminal high K⁺ stimulus, this effect was not statistically significant. While prolonged exposure to tumor-derived factors may influence neuronal excitability, the current experimental design does not specifically address such mechanisms, and these observations should be interpreted within the limitations of the current experimental design. Also, conditioned medium was generated under standardized cell density and confluency without additional protein-based normalization, consistent with common practice in functional assays, although such normalization may further improve inter-assay consistency in future studies.

The use of serum-containing medium (10% FBS) supports neuronal viability following dissociation but may promote glial proliferation during extended culture. In this protocol, functional assays are performed within 24–48 h to minimize glial overgrowth while preserving neuronal responsiveness. For longer culture durations, the use of mitotic inhibitors (e.g., Ara-C) may be considered to limit non-neuronal proliferation, as described previously¹³, although such approaches may alter neuronal phenotype and should be interpreted within the limitations of the experimental design. The inclusion of ATF3 immunostaining provides a complementary molecular readout of neuronal stress and activation responses. Overall, this protocol is broadly applicable to studies of cancer neuroscience, peripheral pain mechanisms, and tumor-induced neuronal plasticity^14,15.

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