Primary Dorsal Root Ganglion Sensory Neuron Culture
Materials Required
Principle
Primary dorsal root ganglion sensory neuron culture isolates DRG neuronal somata from rodent or human ganglia, dissociates tissue enzymatically and mechanically, and maintains post-mitotic sensory neurons in vitro for readouts such as neurite outgrowth, immunocytochemical marker expression, calcium imaging, electrophysiology, RNA/protein analysis, or neuropeptide release assays[1][2][3][6][7]. The method reflects peripheral sensory neuron biology because DRG neurons are primary sensory neurons whose cell bodies reside in dorsal root ganglia and whose cultured dissociated cells can retain neuronal morphology, sensory-neuron marker expression, and stimulus-responsive properties depending on the downstream assay[2][3][4][6][7].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use sterile PBS or HBSS for tissue handling and washing; enzymatic dissociation has been reported with trypsin, collagenase/dispase, collagenase/trypsin, or papain-based systems depending on species, age, and protocol design[1][2][3][4][6][8].
• Use Neurobasal-based medium, B27 supplement, glutamine or GlutaMAX, glucose when specified, antibiotics when specified, and NGF-containing culture medium when the selected protocol requires trophic support[1][2][3][4][6].
• Use poly-D-lysine, poly-L-lysine, laminin, or coated coverslips/plates to support neuronal adhesion and neurite outgrowth when specified by the selected protocol[1][3][4][6][8].
• Use 5-fluorouracil, cytarabine, density-gradient separation, or immunopanning only when neuronal enrichment or suppression/removal of proliferating non-neuronal cells is part of the experimental objective[4][8][9].
• Use neuronal markers such as NF200, neuron-specific enolase, βIII-tubulin, or MAP2 for culture identification when these markers match the assay objective and species/protocol context[1][4][8].
• Use ELISA readouts for neuropeptides such as CGRP or substance P when the culture is being used for neuropeptide-release experiments[2][3].
• Use calcium indicators or genetically encoded calcium reporters when the objective is stimulus-evoked calcium imaging of cultured DRG neurons[1][2][3].
• Use a stereomicroscope and fine dissection tools for vertebral-column exposure, spinal cord removal, ganglion collection, and removal of attached nerve fibers[1][2][5][7].
• Use a tissue-culture hood, CO2 incubator, centrifuge, cell strainer or filter when specified, hemocytometer, coated culture plates or coverslips, and fluorescence or functional imaging equipment matched to the downstream assay[1][2][3][4][7][8].
• Use Neurobasal-based medium, B27 supplement, glutamine or GlutaMAX, glucose when specified, antibiotics when specified, and NGF-containing culture medium when the selected protocol requires trophic support[1][2][3][4][6].
• Use poly-D-lysine, poly-L-lysine, laminin, or coated coverslips/plates to support neuronal adhesion and neurite outgrowth when specified by the selected protocol[1][3][4][6][8].
• Use 5-fluorouracil, cytarabine, density-gradient separation, or immunopanning only when neuronal enrichment or suppression/removal of proliferating non-neuronal cells is part of the experimental objective[4][8][9].
• Use neuronal markers such as NF200, neuron-specific enolase, βIII-tubulin, or MAP2 for culture identification when these markers match the assay objective and species/protocol context[1][4][8].
• Use ELISA readouts for neuropeptides such as CGRP or substance P when the culture is being used for neuropeptide-release experiments[2][3].
• Use calcium indicators or genetically encoded calcium reporters when the objective is stimulus-evoked calcium imaging of cultured DRG neurons[1][2][3].
• Use a stereomicroscope and fine dissection tools for vertebral-column exposure, spinal cord removal, ganglion collection, and removal of attached nerve fibers[1][2][5][7].
• Use a tissue-culture hood, CO2 incubator, centrifuge, cell strainer or filter when specified, hemocytometer, coated culture plates or coverslips, and fluorescence or functional imaging equipment matched to the downstream assay[1][2][3][4][7][8].
Experimental Procedure
• Obtain institutional animal or human tissue approval before tissue collection, select animal age/species according to the intended protocol, and plan the number of animals because DRG neurons are post-mitotic and cultures cannot be expanded by passaging[1][2][7].
• Prepare coated culture surfaces before dissociation; published protocols use poly-lysine-family coatings alone or combined with laminin, and the selected coating should match the cited protocol and downstream assay[1][3][4][6][8].
• Prepare sterile dissociation and culture media before dissection; reported dissociation approaches include trypsin incubation for embryonic rat DRG, collagenase/dispase digestion for murine DRG, prolonged trypsin/collagenase digestion for postnatal/adult rat sensory neurons, and papain-based systems in some mouse primary neuron protocols[2][4][6][8].
• Collect DRG under a stereomicroscope by removing the spinal column or exposing the vertebral canal, removing spinal cord and meninges/dura, isolating ganglia, and trimming attached nerve roots or fibers[1][2][5][7].
• Digest isolated DRG using the enzyme system from the selected protocol; examples include 0.25% trypsin for 30 min at 37°C in embryonic rat DRG, collagenase/dispase digestion in murine protocols, and trypsin/collagenase digestion for postnatal or adult rat sensory neurons[2][4][6].
• Mechanically triturate digested ganglia gently to form a single-cell suspension, avoid bubble formation during pipetting, and use filtration or low-speed centrifugation steps when specified to remove debris and recover cells[2][4][6].
• Plate dissociated cells onto coated plates or coverslips in the reported culture medium; one embryonic rat protocol seeded 1 × 105 cells per well in six-well plates, while mouse protocols scale plating density and culture area according to immunocytochemistry, imaging, western blotting, RNA sequencing, or other downstream applications[1][4].
• Maintain cultures at 37°C in a CO2 incubator and use medium changes or antimitotic treatment only as reported by the selected protocol; one rat protocol replaced medium after 48 h with 5-fluorouracil-containing basal medium and then changed medium every 3 days, whereas other protocols use alternative enrichment approaches or mixed DRG cultures depending on experimental goals[1][2][4][8][9].
• Confirm culture identity by neuronal morphology and marker staining when required; published readouts include NF200 and NSE immunofluorescence or RT-PCR, βIII-tubulin staining, and assay-specific imaging of neurites or neuronal somata[1][4][8].
• For functional assays, measure neuropeptide release by ELISA, stimulus-evoked calcium signals by calcium imaging, electrophysiological responses by patch-clamp, or molecular changes by RNA/protein assays according to the validated downstream application of the chosen culture protocol[1][2][3][6][7].
• Include appropriate positive and negative controls for the downstream assay; published examples include neuropeptide-release stimulation controls, immunocytochemical identification controls, and stimulus-response validation in functional imaging assays[1][2][3][4].
• Prepare coated culture surfaces before dissociation; published protocols use poly-lysine-family coatings alone or combined with laminin, and the selected coating should match the cited protocol and downstream assay[1][3][4][6][8].
• Prepare sterile dissociation and culture media before dissection; reported dissociation approaches include trypsin incubation for embryonic rat DRG, collagenase/dispase digestion for murine DRG, prolonged trypsin/collagenase digestion for postnatal/adult rat sensory neurons, and papain-based systems in some mouse primary neuron protocols[2][4][6][8].
• Collect DRG under a stereomicroscope by removing the spinal column or exposing the vertebral canal, removing spinal cord and meninges/dura, isolating ganglia, and trimming attached nerve roots or fibers[1][2][5][7].
• Digest isolated DRG using the enzyme system from the selected protocol; examples include 0.25% trypsin for 30 min at 37°C in embryonic rat DRG, collagenase/dispase digestion in murine protocols, and trypsin/collagenase digestion for postnatal or adult rat sensory neurons[2][4][6].
• Mechanically triturate digested ganglia gently to form a single-cell suspension, avoid bubble formation during pipetting, and use filtration or low-speed centrifugation steps when specified to remove debris and recover cells[2][4][6].
• Plate dissociated cells onto coated plates or coverslips in the reported culture medium; one embryonic rat protocol seeded 1 × 105 cells per well in six-well plates, while mouse protocols scale plating density and culture area according to immunocytochemistry, imaging, western blotting, RNA sequencing, or other downstream applications[1][4].
• Maintain cultures at 37°C in a CO2 incubator and use medium changes or antimitotic treatment only as reported by the selected protocol; one rat protocol replaced medium after 48 h with 5-fluorouracil-containing basal medium and then changed medium every 3 days, whereas other protocols use alternative enrichment approaches or mixed DRG cultures depending on experimental goals[1][2][4][8][9].
• Confirm culture identity by neuronal morphology and marker staining when required; published readouts include NF200 and NSE immunofluorescence or RT-PCR, βIII-tubulin staining, and assay-specific imaging of neurites or neuronal somata[1][4][8].
• For functional assays, measure neuropeptide release by ELISA, stimulus-evoked calcium signals by calcium imaging, electrophysiological responses by patch-clamp, or molecular changes by RNA/protein assays according to the validated downstream application of the chosen culture protocol[1][2][3][6][7].
• Include appropriate positive and negative controls for the downstream assay; published examples include neuropeptide-release stimulation controls, immunocytochemical identification controls, and stimulus-response validation in functional imaging assays[1][2][3][4].
Troubleshooting
Low adherent neuron number after dissociation:
Possible causeProlonged or overly harsh digestion damaging DRG neurons.
Solution
Restrict digestion to the time reported for the chosen protocol and use gentle trituration; one rat study reported that prolonged digestion reduced adherent cell number and survival time[4].
Poor single-cell dissociation:
Possible causeInsufficient digestion or inadequate mechanical separation.
Solution
Use the protocol-specified enzyme incubation followed by gentle repeated pipetting and low-speed centrifugation rather than extending digestion beyond the validated range[4][6].
Excess non-neuronal cell expansion during culture:
Possible causeProliferation of Schwann cells, fibroblasts, or other non-neuronal cells while mature neurons are post-mitotic.
Solution
Use a literature-supported enrichment strategy such as delayed 5-fluorouracil treatment, density-gradient purification, or immunopanning only when higher neuronal purity is required[4][8][9].
Culture purity is insufficient for bulk RNA or protein assays:
Possible causeThat whole DRG cultures contain neuronal and non-neuronal cells.
Solution
Use neuronal enrichment or purification before bulk molecular analysis when the goal is neuron-specific RNA or protein measurement[1][9].
References:
- [1]. Smith PR, et al. Protocol for the isolation and culture of mouse dorsal root ganglion neurons for imaging applications. STAR Protoc. 2023;4(4):102717. [Content Brief]
- [2]. Perner C, et al. Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release. STAR Protoc. 2021;2(1):100333. [Content Brief]
- [3]. Lin YT, et al. Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release. J Vis Exp. 2018;(140):57569. [Content Brief]
- [4]. Shen H, et al. An integrated cell isolation and purification method for rat dorsal root ganglion neurons. J Int Med Res. 2019;47(7):3253-3260. [Content Brief]
- [5]. Sleigh JN, et al. A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia. BMC Res Notes. 2016;9:82. [Content Brief]
- [6]. Malin SA, et al. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc. 2007;2(1):152-160. [Content Brief]
- [7]. Valtcheva MV, Copits BA, Davidson S, Sheahan TD, Pullen MY, McCall JG, et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc. 2016;11(10):1877-1888. [Content Brief]
- [8]. Katzenell S, et al. Isolation, Purification, and Culture of Primary Murine Sensory Neurons. Methods Mol Biol. 2017;1656:229-251. [Content Brief]
- [9]. Zuchero JB. Purification and culture of dorsal root ganglion neurons. Cold Spring Harb Protoc. 2014;2014(8):826-838. [Content Brief]
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