Optimized primary dorsal root ganglion cell culture protocol for reliable K⁺ current patch-clamp recordings

DRG primary neuron cultures, derived from rodents, closely mimic properties of sensory neurons in vivo and are highly useful for studying pain and neurological disorders. These cultures are pivotal in patch-clamp electrophysiology for sensory neuron properties analysis. A detailed, replicable protocol in scientific research ensures experiment accuracy and reproducibility. This paper provides comprehensive details for replicating the protocol and achieving consistent results in primary DRG cell culture as used for patch-clamp recordings. We outlined a comprehensive protocol for establishing primary DRG cell culture, optimized for improved gigaseal formation in whole-cell patch-clamp recordings. Additionally, we conducted a simulation study focused on recording macroscopic K⁺ channels. The findings established an optimized novel protocol that works reliably for whole-cell patch-clamp recordings and data analysis using primary DRG cells prepared as described in this publication. The details for the protocol in the literature are dispersed across various publications, making it challenging to find a comprehensive summary in one source. This study confirms, for the first time, the efficacy of using fewer protocol steps, which reduces stress and variability in obtaining suitable cells for patch-clamp recordings compared to existing methods in the literature. Given the challenges posed by the dissociation process of primary DRG cells and the importance of comprehensive method documentation in the literature, the protocol presented provides improved and consistent applications of primary DRG cell culture in patch-clamp recordings.