Neuromuscular Junction Formation Assay

Primary myoblasts were isolated from skeletal muscles of P0.5 mouse pups and differentiated into myotubes as previously described [10 (link)]. Myotubes were resuspended in neuronal culture medium and then plated onto coverslips with co-cultured astrocytes and hiMNs at 40 to 50 dpi. After another 4 to 7 days, these sandwich cultures of myotubes, hiMNs, and astrocytes were live-stained with rhodamine-conjugated α-BTX (Invitrogen, 1:10,000) for 1 h at 37 °C. α-BTX labeled cells were then processed for immunostaining with antibodies of SYN1 (Cell Signaling Technology, 1:500) and MHC (Sigma, 1:1000). NMJ formation frequency was presented as the percentage of NMJs on myotubes associated with hiMNs networks.