This study presents a detailed protocol for applying Bioluminescence Resonance Energy Transfer (BRET) assays to primary neuronal cultures derived from mouse embryos. While BRET is widely used for studying G protein-coupled receptor (GPCR) activity in heterologous cell lines, its application in neuronal systems has been limited. The authors describe a method to overcome this limitation by adapting BRET assays for primary cultured neurons. The protocol includes steps for generating plasmid constructs, preparing lentiviruses, plating and transducing neurons, and collecting and analyzing BRET data. The method emphasizes the use of NanoBRET, a variant of BRET that employs NanoLuc® luciferase as the energy donor, offering higher sensitivity and reduced biological interference compared to older systems. This protocol allows for real-time monitoring of GPCR signaling, receptor trafficking, and interactions in a native neuronal environment, which is critical for understanding cell-specific signaling dynamics. The study also provides a comparative analysis of oxytocin receptor interactions in HEK293 cells and neurons, demonstrating the importance of studying these receptors in their native context.

Keywords: BRET, GPCR, primary neuronal cultures, NanoBRET, lentiviral transduction, receptor trafficking

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