Abstract
Cerebrospinal fluid-contacting neurons (CSF-cNs) are a specialized group of multifunctional neurons located around the central canal of the spinal cord. They play critical roles in motor regulation, postural maintenance, and spinal cord injury repair. However, the molecular mechanisms underlying the multifunctionality of CSF-cNs remain poorly understood, partly due to the lack of established in vitro methods for their efficient selection and purification, which significantly hinders mechanistic investigations. In this study, we describe a standardized method using a PKD2L1 promoter-driven lentiviral system, which enables effective enrichment and identification of CSF-cNs in vitro through GFP labeling and puromycin selection. This protocol includes key steps such as construction of the PKD2L1 promoter-driven lentiviral vector, spinal cord tissue collection and digestion from neonatal mice, lentiviral infection, antibiotic selection, and immunofluorescence-based identification of CSF-cNs. Our method provides a reliable platform for obtaining high-purity CSF-cNs (>99%), which facilitates their functional and mechanistic studies for regenerative approaches in vitro. Key features • Enables specific labeling and selection of CSF-cNs using a constructed PKD2L1 promoter-driven lentiviral vector. • Combines GFP-based fluorescence tracing and puromycin selection for efficient dual-mode enrichment of high-purity CSF-cNs. • Provides a simple and reproducible workflow that facilitates in vitro isolation and validation of CSF-cNs for disease modeling and spinal cord injury repair.