NanoBiT Luciferase-based live-cell assay for cell–cell fusion quantification

To quantify cell–cell fusion, we adapted the NanoBiT technology based on two-subunit NanoLuc luciferase (Nano-Glo® HiBiT system, Promega). DF-1/HiBiT cells were seeded in a 6-well plate (0.45 × 10⁶ cells/well) and after 24 h transfected with 2.5 µg of pMCAS(3Flag-Sync1-MS)dsRed. Forty-eight hours after transfection, the DF-1/HiBiT-Sync1 cells were mixed with DF-1/LgBiT/FuTraP cells in ratios 2 × 10⁴: 1 × 10⁴ cells, respectively, and transferred in triplicate to a whole-white 96-well plate (Costar). For inhibition of cell–cell fusion, the mixture of DF-1/HiBiT-Sync1 and DF-1/LgBiT/pFuTraP cells was seeded in a whole-white 96-well plate and cultured in the medium containing either sS1 (as well as RCASBP(B)) or RCASBP(B)GFP alone. After 24 h incubation, the supernatant from the cells was replaced with 100 µl of OptiMEM medium and the cells were incubated for additional 60 min. Afterwards, 20 µl of 37 °C-equilibrated Nano-Glo Live Cell Reagent (19 µl of LCS Dilution Buffer and 1 µl of Live Cell Substrate; Promega) containing the luciferase substrate, furimazine, was added to cells and the plate was placed on an orbital shaker at 300 rpm, 15 s. At this point, reassorted HiBiT-LgBiT fragments in the fused cells started to oxidise the substrate, resulting in luminescence emission. The relative luminescence was measured in an EnVision Plate Reader (PerkinElmer) immediately after adding the Live Cell Reagent.

To verify the surface expression of 3Flag-Sync1-MS, 2.5 × 10⁵ living cells were immunostained 48 h after the transfection with monoclonal Anti-Flag® M2-FITC antibody (Sigma, 1:1000 dilution in PBS with 2% calf serum; 1 ml per 10⁶ cells) and evaluated by flow cytometry. A live gate was created according to Hoechst 33258 staining. Transfection efficiency was assessed by flow cytometry according to the dsRed fluorescence (Additional file 8: Fig. S7).