培养成年小鼠背根神经节原代神经元，用于评估用肿瘤细胞条件培养基治疗后的神经元功能
Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with Tumor Cell-Conditioned Medium.
摘要(中文译文)
感觉神经元与肿瘤细胞之间的相互作用日益被认为是肿瘤进展和癌症相关疼痛的关键促成因素。在此，我们描述了一种可重复的方案，用于成年小鼠背根神经节（DRG）神经元的分离和培养，随后通过功能和分子检测评估其对肿瘤来源分泌因子的反应。解剖成年DRG，使用Liberase DH进行酶消化，并解离为单神经元。将细胞接种于层粘连蛋白包被的盖玻片上，并维持于限定培养条件中，以促进神经元存活同时尽量减少胶质细胞过度生长。随后，将培养的神经元暴露于肿瘤细胞条件培养基，以模拟神经元-肿瘤界面的旁分泌信号传导。通过使用荧光指示剂的活细胞钙成像监测功能反应，从而能够量化神经元活动和刺激特异性反应性。同时，通过ATF3免疫染色评估神经元应激和损伤。总之，这些互补方法为研究肿瘤分泌因子如何致敏或激活感觉神经元提供了强有力的平台。该方案将成年DRG培养、肿瘤条件培养基处理、钙成像和分子读数整合到单一工作流程中，使研究人员能够剖析神经元-肿瘤通讯的机制。该方法广泛适用于癌症神经科学、疼痛信号传导以及鉴定在疾病背景下调节神经元功能的分泌介质的研究。
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.
如何引用
AMA
Khalil Ali Ahmad Kasm, Sandeep Dembla, Angela C Hirbe, Andrew J Shepherd, Yuan Pan. Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with Tumor Cell-Conditioned Medium.. Journal of visualized experiments : JoVE. 2026; doi:10.3791/70748.
APA
Khalil Ali Ahmad Kasm, Sandeep Dembla, Angela C Hirbe, Andrew J Shepherd, Yuan Pan (2026). Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with Tumor Cell-Conditioned Medium.. Journal of visualized experiments : JoVE. https://doi.org/10.3791/70748
GB/T 7714
Khalil Ali Ahmad Kasm, Sandeep Dembla, Angela C Hirbe, Andrew J Shepherd, Yuan Pan. Culturing Primary Adult Mouse Dorsal Root Ganglion Neurons for Assessing Neuronal Function Following Treatment with Tumor Cell-Conditioned Medium.[J]. Journal of visualized experiments : JoVE, 2026 doi:10.3791/70748.
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