SARS-CoV-2 Membrane Fusion Assay

The DSP assay using 293FT cells was performed as described previously (Yamamoto et al., 2020 (link)) to monitor SARS-CoV-2-S-mediated membrane fusion. Briefly, effector cells expressing SARS-CoV-2-S protein with DSP8-11, target cells expressing ACE2, and transmembrane serine protease 2 (TMPRSS2) with DSP1-7 were seeded in 10 cm culture dishes (4×10⁶ cells/10 mL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 µM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each peptide dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 µL of each single-cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Fisher Scientific, Waltham, MA, USA). After incubation at 37°C for 4 h, luciferase activity was measured using a Centro xS960 luminometer (Berthold, Bad-Wildbad, Germany).