Protocol for stable cell line production to express muscle-type nicotinic receptor

The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at quantities sufficient for single-particle cryoelectron microscopy (cryo-EM) and may be applicable to other hetero-multimeric protein complexes. For complete details on the use and execution of this protocol, please refer to Li et al.¹.