Figure 1.

Syncytia assay system based on split NanoLuc. (A) Diagram of 293T coculture system for syncytia detection. Fused cells of 293T‐ACE2/LgBiT and 293T‐spike/HiBiT lead to complementation of NanoLuc, which can be detected with Nano‐Glo live cell reagent. (B) Optimization of protein combination for efficient syncytia detection. 293T cells transfected to express the indicated proteins were cocultured and luminescence was measured 12 h later. (C) Luminescences of coculture cells expressing ACE2/LgBiT and spike/HiBiT (S + ACE2), ACE2/LgBiT and HiBiT (No spike) or LgBiT and spike/HiBiT(No ACE2) were determined at indicated time points. (D) Real‐time measurement of luminescences of coculture cells as described in (C) with extended Nano‐Glo substrate (Endurazine). Inset, enlargement of the first 2 h. (E) Represented images of syncytia formed at 8 h (upper panel) or 24 h (lower panel) of coculture cells as described in (C). Bar, 20 μm. (F) Cell viability of cocultured cells as described above were measured at the indicated time points with Cell‐Titer Glo. (G) Cells transfected with increasing amount of ACE2 and 0.4 μg of LgBiT plasmid were cocultured with fixed amount of spike/HiBiT cells and luminescences were measured at 8 h post‐coculture. ACE2 expression was immunoblotted. (H) Cells transfected with increasing amount of spike and 0.4 μg of HiBiT plasmid were cocultured with fixed amount of ACE2/LgBiT cells and luminescences were measured at 8 h post‐coculture. Spike expression was immunoblotted.